U.S. patent number 11,214,768 [Application Number 15/554,963] was granted by the patent office on 2022-01-04 for methods of generating functional human tissue.
This patent grant is currently assigned to PRESIDENT AND FELLOWS OF HARVARD COLLEGE. The grantee listed for this patent is PRESIDENT AND FELLOWS OF HARVARD COLLEGE. Invention is credited to George M. Church, Kimberly A. Homan, David B. Kolesky, Jennifer A. Lewis, Alex H. M. Ng, Mark A. Skylar-Scott.
United States Patent |
11,214,768 |
Lewis , et al. |
January 4, 2022 |
Methods of generating functional human tissue
Abstract
Methods of tissue engineering, and more particularly methods and
compositions for generating various vascularized 3D tissues, such
as 3D vascularized embryoid bodies and organoids are described.
Certain embodiments relate to a method of generating functional
human tissue, the method comprising embedding an embryoid body or
organoid in a tissue construct comprising a first vascular network
and a second vascular network, each vascular network comprising one
or more interconnected vascular channels; exposing the embryoid
body or organoid to one or more biological agents, a biological
agent gradient, a pressure, and/or an oxygen tension gradient,
thereby inducing angiogenesis of capillary vessels to and/or from
the embryoid body or organoid; and vascularizing the embryoid body
or organoid, the capillary vessels connecting the first vascular
network to the second vascular network, thereby creating a single
vascular network and a perfusable tissue structure.
Inventors: |
Lewis; Jennifer A. (Cambridge,
MA), Skylar-Scott; Mark A. (Brookline, MA), Kolesky;
David B. (Cambridge, MA), Homan; Kimberly A.
(Somerville, MA), Ng; Alex H. M. (Cambridge, MA), Church;
George M. (Brookline, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
PRESIDENT AND FELLOWS OF HARVARD COLLEGE |
Cambridge |
MA |
US |
|
|
Assignee: |
PRESIDENT AND FELLOWS OF HARVARD
COLLEGE (Cambridge, MA)
|
Family
ID: |
1000006031990 |
Appl.
No.: |
15/554,963 |
Filed: |
March 3, 2016 |
PCT
Filed: |
March 03, 2016 |
PCT No.: |
PCT/US2016/020601 |
371(c)(1),(2),(4) Date: |
August 31, 2017 |
PCT
Pub. No.: |
WO2016/141137 |
PCT
Pub. Date: |
September 09, 2016 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20180030409 A1 |
Feb 1, 2018 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
62294118 |
Feb 11, 2016 |
|
|
|
|
62250338 |
Nov 3, 2015 |
|
|
|
|
62127549 |
Mar 3, 2015 |
|
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
5/0656 (20130101); C12N 5/0697 (20130101); B29C
64/106 (20170801); C12N 5/0618 (20130101); A61L
27/38 (20130101); C12N 5/0062 (20130101); C12N
5/0619 (20130101); A61K 35/545 (20130101); C12N
5/069 (20130101); C12N 2535/00 (20130101); B29K
2089/00 (20130101); C12N 2502/45 (20130101); B29K
2105/0061 (20130101); B33Y 70/00 (20141201); C12N
2533/54 (20130101); C12N 2501/727 (20130101); C12N
2501/60 (20130101); C12N 2506/45 (20130101); C12N
2501/165 (20130101); C12N 2502/28 (20130101); B33Y
80/00 (20141201); C12N 2501/40 (20130101); B33Y
10/00 (20141201); C12N 2533/30 (20130101) |
Current International
Class: |
C12N
5/00 (20060101); A61K 35/545 (20150101); A61L
27/38 (20060101); C12N 5/071 (20100101); B29C
64/106 (20170101); C12N 5/079 (20100101); C12N
5/077 (20100101); C12N 5/0793 (20100101); B33Y
10/00 (20150101); B33Y 70/00 (20200101); B33Y
80/00 (20150101) |
Field of
Search: |
;435/11 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
101410507 |
|
Apr 2009 |
|
CN |
|
101410507 |
|
Apr 2009 |
|
CN |
|
101534747 |
|
Sep 2009 |
|
CN |
|
106163581 |
|
Nov 2016 |
|
CN |
|
2 762 558 |
|
Aug 2014 |
|
EP |
|
2009-531067 |
|
Sep 2009 |
|
JP |
|
WO 02/078439 |
|
Oct 2002 |
|
WO |
|
WO 2007/112192 |
|
Oct 2007 |
|
WO |
|
WO 2008/008229 |
|
Jan 2008 |
|
WO |
|
WO 2012/036225 |
|
Mar 2012 |
|
WO |
|
WO 2013/096741 |
|
Jun 2013 |
|
WO |
|
WO 2014/090993 |
|
Jun 2014 |
|
WO |
|
WO 2014/168719 |
|
Oct 2014 |
|
WO |
|
WO 2016/141137 |
|
Sep 2016 |
|
WO |
|
Other References
Mohammadi, M. H. et al., Skin diseases modeling using combined
tissue engineering and microfluidic technologies. Advanced
Healthcare Materials, 2016 (published online: Aug. 22, 2016), vol.
5, Issue 19, pp. 2459-2480. (Year: 2016). cited by examiner .
International Search Report and Written Opinion received in PCT
Application No. PCT/US2016/020601, dated May 31, 2016. cited by
applicant .
Mondrinos et al., "Engineering De Novo Assembly of Fetal Pulmonary
Organoids," Tissue Engineering, Part A, 20(21-22):2892-2907 (Jun.
25, 2014). cited by applicant .
Takebe et al., "Generation of Functional Human Vascular Network,"
Transplantation Proceedings, 44(4):1130-1133 (May 1, 2012). cited
by applicant .
Takebe et al., "Engineering of Human Hepatic Tissue with Functional
Vascular Networks," Organogenesis, 10(2):260-267 (Jan. 22, 2014).
cited by applicant .
Extended European Search Report (EESR) with the supplementary
European search report and the European search opinion received for
the corresponding European Application No. 16759465.4 dated Oct. 5,
2018. cited by applicant .
Notification Concerning Transmittal of International Preliminary
Report on Patentability dated Sep. 14, 2017, International
Preliminary Report on Patentability and Written Opinion of the
International Searching Authority received in PCT Application No.
PCT/US2016/020601. cited by applicant .
Reporting Letter received from Japanese associate dated Jan. 31,
2020 enclosing English translation of Official Action received in
Japanese Application No. JP 2017-546660 dated Jan. 2, 2020
(Japanese version included). cited by applicant .
Reporting Letter received from Japanese associate dated Jan. 21,
2020 and Official Action received in Japanese Application No. JP
2017-546660 dated Jan. 2, 2020 (in Japanese). cited by applicant
.
Hosoe, H., et al., "Investigation of VEGF and PDGF signals in
vascular formation by 3D culture models using mouse ES cells," Stem
Cell Discovery, 2(2):70-77 (2012). cited by applicant .
Brownfield, D., et al., "Patterned Collagen Fibers Orient Branching
Mammary Epithelium through Distinct Signaling Modules," Current
Biology, 23:703-709 (2013). cited by applicant .
Kolesky, D., et al., "3D Bioprinting of Vascularized, Heterogeneous
Cell-Laden Tissue Constructs," Advanced Materials, 26:3124-3130
(2014). cited by applicant .
Reporting Letter received from European associate dated Jan. 20,
2020 and Communication Pursuant to Article 94(3) received in
European Application No. 16759465.4 dated Jan. 14, 2020. cited by
applicant .
Reporting letter dated Jul. 7, 2020 enclosing First Office Action
received in Chinese Application No. 201680022237.7 dated Jun. 15,
2020 (in Chinese and including English translation of the First
Office Action). cited by applicant .
Examination Report received in the corresponding European Patent
Application No. 16759465.4 dated Jan. 18, 2021, and a letter from
the European associate dated Jan. 26, 2021 reporting the
Examination Report. cited by applicant .
Bertassoni, L.E., et al., "Hydrogel bioprinted microchannel
networks for vascularization of tissue engineering contructs," Lab
Chip, 14:2202-2211 (2014). cited by applicant .
Lee, V.K., et al., "Construction of 3D Tissue with Perfused Vessels
and Capillaries through 3D Bio-Printing," 40.sup.th Annual
Northeast Bioengineering Conference, 2 pgs. (2014). cited by
applicant .
Reporting Letter dated Jan. 27, 2021 enclosing the Notification of
Second Office Action received in Chinese Patent Application No.
201680022237.7 dated Jan. 25, 2021 (in Chinese and including
English translation of the Second Office Action). cited by
applicant .
Examination Report No. 2 received in the corresponding Australian
Patent Application No. 2016226178 dated Mar. 31, 2021 and a letter
from the Australian associate dated Apr. 14, 2021 reporting the
Examination Report. cited by applicant .
Reporting Letter dated Sep. 1, 2021 enclosing the Rejection
Decision received in Chinese Application No. 201680022237.7 dated
Aug. 18, 2021 (in Chinese and including English translation of the
Rejection Decision). cited by applicant .
First Examination Report received in the corresponding Australian
Patent Application No. 2016226178 dated Aug. 25, 2020, and a copy
of a letter from the Australian associate dated Sep. 2, 2020
reporting the First Examination Report. cited by applicant.
|
Primary Examiner: Bertoglio; Valarie E
Attorney, Agent or Firm: Crowell & Moring LLP
Government Interests
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with Government support under Grant No.
IRMIHG008525-01, awarded by the National Institute of Health (NIH).
The Government has certain rights in this invention.
Parent Case Text
RELATED APPLICATIONS
The present patent document is a .sctn. 371 filing based on PCT
Application Serial No. PCT/US2016/020601, filed Mar. 3, 2016, which
claims the benefit of the filing date under 35 U.S.C. .sctn. 119(e)
of Provisional U.S. Patent Application Ser. No. 62/127,549, filed
Mar. 3, 2015; Provisional U.S. Patent Application Ser. No.
62/250,338, filed Nov. 3, 2015; and U.S. Patent Application Ser.
No. 62/294,118, filed Feb. 11, 2016, which are hereby incorporated
by reference.
Claims
The invention claimed is:
1. An in vitro method of generating functional human tissue, the
method comprising: (a) embedding an embryoid body or organoid in an
in vitro tissue construct, the tissue construct comprising: (i) a
first vascular network comprising one or more interconnected
vascular channels, and (ii) a second vascular network comprising
one or more interconnected vascular channels; (b) exposing the
embedded embryoid body or organoid to one or more of a biological
agent gradient, a pressure gradient, and/or an oxygen tension
gradient, thereby inducing angiogenesis of capillary vessels to
and/or from the embryoid body or organoid via delivery of the
gradient by at least one of the first and the second vascular
networks; and (c) wherein exposing the embedded embryoid body or
organoid to one or more of a biological agent gradient, a pressure
gradient, and/or an oxygen tension gradient promotes vascularizing
the embryoid body or organoid, the capillary vessels connecting the
first vascular network to the second vascular network, thereby
creating functional human tissue having a single vascular network
and a perfusable tissue structure.
2. The method of claim 1, wherein the biological agent gradient
includes one or more of the following: growth factors, morphogens,
small molecules, drugs, hormones, DNA, shRNA, siRNA, nanoparticles,
mRNA, modified mRNA.
3. The method of claim 1, wherein the one or more interconnected
vascular channels are formed by a manufacturing process or by a
biological developmental process that includes at least one of
vasculogenesis, angiogenesis, or tubulogenesis.
4. The method of claim 1, wherein the one or more of biological
agent gradient, the pressure gradient, and/or the oxygen tension
gradient further direct development, differentiation, and/or
functioning of the embryoid body or organoid.
5. The method of claim 1, wherein the first vascular network and
the second vascular network are independently addressable.
6. The method of claim 1, wherein the first vascular network and
the second vascular network are not in contact with each other
prior to the vascularizing step (c).
7. The method of claim 1, wherein the first vascular network
comprises an arterial plexus and the second vascular network
comprises a venous plexus.
8. The method of claim 1, wherein the single vascular network
comprises at least one of an interpenetrating vascular network or a
branched interpenetrating vascular network.
9. The method of claim 1, wherein the single vascular network
comprises interconnected arterial and venous channels.
10. The method of claim 1, wherein the embryoid body or organoid is
created by culturing at least one of: pluripotent stem cells,
multipotent stem cells, progenitor cells, terminally differentiated
cells, endothelial cells, endothelial progenitor cells,
immortalized cell lines, or primary cells.
11. The method of claim 1, wherein, prior to, during and/or after
the embedding, the embryoid body or organoid is further
differentiated into a tissue containing at least one of pluripotent
stem cells, multipotent stem cells, progenitor cells, terminally
differentiated cells, endothelial cells, endothelial progenitor
cells, immortalized cell lines, or primary cells.
12. The method of claim 1, wherein the embryoid body or organoid is
selected from the group consisting of: cerebral organoid, thyroid
organoid, intestinal or gut organoid, hepatic organoid, pancreatic
organoid, gastric organoid, kidney organoid, retinal organoid,
cardiac organoid, bone organoid, and epithelial organoid.
13. The method of claim 1, wherein the embryoid body or organoid is
exposed to the biological agent gradient by at least one of:
diffusion of one or more biological agents within the tissue
construct; localized deposition of materials loaded with one or
more biological agents within the tissue construct; localized
de-novo production of growth factors by localized protein
translation; or perfusion of one or both of the first and second
vascular networks with one or more biological agents.
14. The method of claim 1, wherein only one of the first and second
vascular networks is exposed to a biological agent gradient prior
to the vascularizing step (c).
15. The method of claim 1, wherein both the first and second
vascular networks are exposed to a biological agent gradient, and
wherein the biological agent concentration in the first vascular
network is different than the biological agent concentration in the
second vascular network.
16. The method of claim 1, wherein both the first and second
vascular networks are exposed to a biological agent gradient, and
wherein the biological agent concentration in the first vascular
network is the same as the biological agent concentration in the
second vascular network.
17. The method of claim 1, wherein the biological agent in the
biological agent gradient comprises is one or more of: vascular
endothelial growth factor (VEGF), basic fibroblast growth factor
(bFGF), sphingosine-1-phosphate (S1P), phorbol myristate acetate
(PMA), hepatocyte growth factor (HGF), monocyte chemotactic
protein-1 (MCP-1), the angiopoietin ANG-1, the angiopoietin ANG-2,
transforming growth factor beta (TGF-.beta.), epidermal growth
factor (EGF), human growth factor, matrix metalloproteinases
(MMP's), or histamine.
18. The method of claim 1, wherein an oxygen partial pressure
gradient is introduced to one or both of the first and second
vascular networks during the exposing step.
19. The method of claim 18, wherein the oxygen partial pressure
gradient is formed by introducing deoxygenated media into one of
the first and second vascular networks, and by introducing
oxygenated media into the other of the first and second vascular
networks.
20. The method of claim 13, wherein one or both of the first and
second vascular networks are subjected to a transmural pressure
during the perfusion.
21. The method of claim 1, wherein, prior to embedding the embryoid
body or organoid in the tissue construct, the embryoid body or
organoid is encapsulated in an extracellular matrix material
comprising a gel.
22. The method of claim 1, wherein the embryoid body or organoid
comprises a first population of embryoid body or organoid cells and
a second population of embryoid body or organoid cells.
23. The method of claim 22, wherein the embryoid body or organoid
comprises at least two of: pluripotent stem cells, multipotent stem
cells, progenitor cells, terminally differentiated cells,
endothelial cells, endothelial progenitor cells, immortalized cell
lines, neural cells, primary cells, or a combination thereof.
24. The method of claim 1, wherein the embryoid body or organoid is
created by: culturing a wild-type population of cells and a
genetically-engineered inducible population of cells in a medium;
inducing direct differentiation and/or transdifferentiation of the
genetically-engineered inducible population of cells into a first
population of the embryoid body or organoid cells; inducing
differentiation of the wild-type population of cells into a second
population of the embryoid body or organoid cells; and thereby
forming the embryoid body or organoid comprising at least the first
population of the embryoid body or organoid cells and the second
population of embryoid body or organoid cells.
25. The method of claim 24, wherein the embryoid body or organoid
is selected from the group consisting of: cerebral organoid,
thyroid organoid, intestinal or gut organoid, hepatic organoid,
pancreatic organoid, gastric organoid, kidney organoid, retinal
organoid, cardiac organoid, bone organoid, and epithelial
organoid.
26. The method of claim 24, wherein the genetically-engineered
inducible population of cells is created by introducing a DNA
delivery element comprising at least one of constitutive promoter,
small molecule inducible promoter, cell-autonomous promoter, cell
non-autonomous promoter, selection marker, or a combination
thereof.
27. The method of claim 24, wherein the first population of the
embryoid body or organoid cells comprises pluripotent stem cells,
multipotent stem cells, progenitor cells, terminally differentiated
cells, endothelial cells, endothelial progenitor cells,
immortalized cell lines, or primary cells.
28. The method of claim 24, wherein the step of inducing direct
differentiation and/or transdifferentiation of the
genetically-engineered inducible population of cells comprises
introducing at least one cue selected from the group consisting of
transcription factors, drugs, small molecules, growth factors,
morphogens, hormones, DNA, shRNA, siRNA, nanoparticles, mRNA,
modified mRNA, heat, light, and mechanical force.
29. The method of claim 24, wherein the induced direct
differentiation and or transdifferentiation is accompanied by a
secondary gene induction.
30. The method of claim 24, wherein the step of culturing is in a
differentiation medium, and wherein the differentiation medium
comprises doxycycline (DOX).
31. The method of claim 24, wherein the wild-type population of
cells comprises induced pluripotent stem cells (iPSCs) or
iPSCs-derived patient-specific cell lines.
32. The method of claim 1, wherein one or both of the first and
second vascular networks comprise microfluidic channels.
33. The method of claim 1, wherein a plurality of the embryoid
bodies or organoids are embedded in the tissue construct.
34. The method of claim 33, wherein the embryoid bodies or
organoids comprise different phenotypes.
35. The method of claim 33, wherein the embryoid bodies or
organoids comprise the same phenotype.
36. The method of claim 1, wherein the tissue construct comprises
an array of the tissue constructs, wherein the embedding, exposing
and vascularizing is carried out in each tissue construct.
37. An implantable, functional human tissue formed by the in vitro
method of claim 1, wherein the embryoid body or organoid is
prepared for an immunofluorescence protocol.
Description
All patents, patent applications and publications, and other
literature references cited herein are hereby incorporated by
reference in their entirety. The disclosures of these publications
in their entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art as
known to those skilled therein as of the date of the invention
described and claimed herein.
BACKGROUND
The ability to create three-dimensional (3D) vascularized tissues
on demand could enable scientific and technological advances in
tissue engineering, drug screening, toxicology, 3D tissue culture,
and organ repair. To produce 3D engineered tissue constructs that
mimic natural tissues and, ultimately, organs, several key
components--cells, extracellular matrix (ECM), and vasculature--may
need to be assembled in complex arrangements. Each of these
components plays a vital role: cells are the basic unit of all
living systems, ECM provides structural support, and vascular
networks provide efficient nutrient and waste transport,
temperature regulation, delivery of factors, and long-range
signaling routes. Without perfusable vasculature within a few
hundred microns of each cell, three-dimensional tissues may quickly
develop necrotic regions. The inability to embed vascular networks
in tissue constructs has hindered progress on 3D tissue engineering
for decades.
Classical experiments performed half a century ago demonstrated the
immense self-organizing capacity of vertebrate cells. Even after
complete dissociation, cells can reaggregate and reconstruct the
original architecture of an organ. More recently, this outstanding
feature was used to rebuild organ parts or even complete organs
from tissue or embryonic stem cells. Such stem cell-derived
three-dimensional cultures are called organoids. Because organoids
can be grown from human stem cells and from patient-derived induced
pluripotent stem cells, they have the potential to model human
development and disease and in a tree-dimensional, biomimetic
environment (Lancaster M A, et al., Cerebral organoids model human
brain development and microcephaly. Nature 501 (7467):373-9
(2013)). Furthermore, they have potential for drug testing and even
future organ replacement strategies (Lancaster et al., 2013). The
organoids are often developed in spinning bioreactors.
New methods of creating embryoid bodies or organoids and tissue
constructs suitable for studies of tissue development and disease,
as well as transplantation are desired.
SUMMARY
Methods of tissue engineering, and more particularly methods and
compositions for generating various vascularized 3D tissues, such
as 3D vascularized embryoid bodies and organoids are described.
Certain embodiments relate to a method of generating functional
human tissue, the method comprising embedding an embryoid body or
organoid in a tissue construct comprising a first vascular network
and a second vascular network, each vascular network comprising one
or more interconnected vascular channels; exposing the embryoid
body or organoid to one or more biological agents, a biological
agent gradient, a pressure, and/or an oxygen tension gradient,
thereby inducing angiogenesis of capillary vessels to and/or from
the embryoid body or organoid; and vascularizing the embryoid body
or organoid, the capillary vessels connecting the first vascular
network to the second vascular network, thereby creating a single
vascular network and a perfusable tissue structure. The one or more
biological agents include one or more of growth factors,
morphogens, small molecules, drugs, hormones, DNA, shRNA, siRNA,
nanoparticles, mRNA, and modified mRNA. The one or more
interconnected vascular channels may be formed by a manufacturing
process or by a biological developmental process that may include
at least one of vasculogenesis, angiogenesis, or tubulogenesis. The
one or more biological agents, the biological agent gradient, the
pressure, and/or the oxygen tension gradient may further direct
development, differentiation, and/or functioning of the embryoid
body or organoid. The first vascular network and the second
vascular network may be independently addressable. The first
vascular network and the second vascular network may not be in
contact with each other. The first vascular network may comprise an
arterial plexus and the second vascular network may comprise a
venous plexus. The single vascular network may comprise an
interpenetrating vascular network and/or a branched
interpenetrating vascular network. The single vascular network may
comprise interconnected arterial and venous channels. The embryoid
body or organoid may be created by culturing at least one of:
pluripotent stem cells, multipotent stem cells, progenitor cells,
terminally differentiated cells, endothelial cells, endothelial
progenitor cells, immortalized cell lines, or primary cells. The
embryoid body or organoid may be created by culturing pluripotent
or multipotent stem cells. The culturing may take place on a
low-adhesion substrate, via a hanging drop method, via aggregation
in microwells, via aggregation in microchannels, or by using a
spinning bioreactor. In the method, prior to, during and/or after
the embedding, the embryoid body or organoid may be further
differentiated into a tissue containing at least one of pluripotent
stem cells, multipotent stem cells, progenitor cells, terminally
differentiated cells, endothelial cells, endothelial progenitor
cells, immortalized cell lines, or primary cells. The embryoid body
or organoid may be a cerebral organoid, thyroid organoid,
intestinal or gut organoid, hepatic organoid, pancreatic organoid,
gastric organoid, kidney organoid, retinal organoid, cardiac
organoid, bone organoid, cancer organoid, or epithelial organoid.
The embryoid body or organoid may be exposed to the one or more
biological agents and/or the biological agent gradient due to
diffusion of the one or more biological agents within the tissue
construct. Alternatively or in addition, the embryoid body or
organoid may be exposed to the one or more biological agents and/or
the biological agent gradient by localized deposition of materials
loaded with the one or more biological agents within the tissue
construct. Alternatively or in addition, the embryoid body or
organoid may be exposed to the one or more biological agents and/or
the biological agent gradient by localized de-novo production of
growth factors by localized protein translation. Alternatively or
in addition, the embryoid body or organoid may be exposed to the
one or more biological agents and/or the biological agent gradient
via perfusion of one or both of the first and second vascular
networks with the one or more biological agents. In the method,
only one of the first and second vascular networks may be perfused
with the one or more biological agents. Alternatively, both the
first and second vascular networks may be perfused with the one or
more biological agents, and a biological agent concentration in the
first vascular network is different than a biological agent
concentration in the second vascular network. Alternatively, both
the first and second vascular networks are perfused with the one or
more biological agents, and a biological agent concentration in the
first vascular network is the same as a biological agent
concentration in the second vascular network. The biological agents
may include one or more of the following growth factors or small
molecules: vascular endothelial growth factor (VEGF), basic
fibroblast growth factor (bFGF), sphingosine-1-phosphate (S1P),
phorbol myristate acetate (PMA), hepatocyte growth factor (HGF),
monocyte chemotactic protein-1 (MCP-1), the angiopoietin ANG-1, the
angiopoietin ANG-2, transforming growth factor beta (TGF-.beta.),
epidermal growth factor (EGF), human growth factor, matrix
metalloproteinases (MMP's), doxycycline, and histamine. In the
method, an oxygen partial pressure gradient may be introduced to
one or both of the first and second vascular networks during
perfusion. The oxygen partial pressure gradient may be formed by
introducing deoxygenated media into one of the first and second
vascular networks, and by introducing oxygenated media into the
other of the first and second vascular networks. The media may be
deoxygenated using either continuous bubbling of nitrogen gas
through media, and/or by adding the enzymes glucose oxidase and
catalase in the presence of glucose. The perfusion may be carried
out at a flow rate of from about 1 microliter per minute to about 1
liter per minute. In the method, one or both of the first and
second vascular networks may be subjected to a transmural pressure
during the perfusion. In the method, prior to embedding the
embryoid body or organoid in the tissue construct, the embryoid
body or organoid may be encapsulated in an extracellular matrix
material. The extracellular matrix material may comprise a gel. In
the method, the embryoid body or organoid may comprise a first
population of embryoid body or organoid cells and a second
population of embryoid body or organoid cells, where the embryoid
body or organoid may comprise at least two of: pluripotent stem
cells, multipotent stem cells, progenitor cells, terminally
differentiated cells, endothelial cells, endothelial progenitor
cells, immortalized cell lines, neural cells, primary cells, or a
combination thereof.
In certain embodiments, the embryoid body or organoid may be
created by culturing a wild-type population of cells and a
genetically-engineered inducible population of cells in a medium,
inducing direct differentiation and/or transdifferentiation of the
genetically-engineered inducible population of cells into the first
population of the embryoid body or organoid cells, inducing
differentiation of the wild-type population of cells into the
second population of the embryoid body or organoid cells, and
thereby forming the embryoid body or organoid comprising at least
the first population of the embryoid body or organoid cells and the
second population of embryoid body or organoid cells. The
genetically-engineered inducible population of cells may be created
by introducing a DNA delivery element comprising at least one of
constitutive promoter, small molecule inducible promoter,
cell-autonomous promoter, cell non-autonomous promoter, selection
marker, or a combination thereof. The first population of the
embryoid body or organoid cells may comprise pluripotent stem
cells, multipotent stem cells, progenitor cells, terminally
differentiated cells, endothelial cells, endothelial progenitor
cells, immortalized cell lines, or primary cells. The second
population of the embryoid body or organoid cells may comprise
neural progenitor cells, where the neural progenitor cells can form
at least one of excitatory neurons, inhibitory interneurons, motor
neurons, dopaminergic neurons, pain receptor neurons, astrocytes,
oligodendrocyte progenitor cells, and/or oligodendrocytes. The step
of inducing direct differentiation and/or transdifferentiation of
the genetically-engineered inducible population of cells may
comprise introducing at least one cue selected from the group
consisting of transcription factors, drugs, small molecules, growth
factors, morphogens, hormones, DNA, shRNA, siRNA, nanoparticles,
mRNA, modified mRNA, heat, light, and mechanical force. The induced
direct differentiation and or transdifferentiation may be
accompanied by a secondary gene induction. The secondary gene
induction may be via providing at least one cue selected from the
group consisting of transcription factors, drugs, small molecules,
growth factors, morphogens, hormones, DNA, shRNA, siRNA,
nanoparticles, mRNA, modified mRNA, heat, light, and mechanical
force. The cue selected for the secondary gene induction may be the
same as the cue selected for the step of inducing direct
differentiation and/or transdifferentiation of the
genetically-engineered inducible population of cells, or may be
different. The first population of the embryoid body or organoid
cells can undergo further development due to induction of a
secondary gene. The induction of the secondary gene may induce an
expression of a constitutively-active protein kinase C (PKC)
protein thereby enhancing at least one of an endothelial sprouting
behavior of the first population of the embryoid body or organoid
cells and neurite outgrowth. The first population of the embryoid
body or organoid cells is endothelial cells. The step of culturing
can take place on a low-adhesion substrate, via a hanging drop
method, via aggregation in microchannels, via aggregation in
microwells, or by using a spinning bioreactor. The ratio of the
first population the embryoid body or organoid cells to the second
population of the embryoid body or organoid cells may be 1:1. Other
ratios are also considered (e.g., 1:2, 1:3, 1:4, etc., 2:1, 3:1,
4:1, etc.). The step of culturing may be in a differentiation
medium. The differentiation medium may comprise doxycycline (DOX)
or other drugs associated with drug-inducible promoters. The one or
more interconnected vascular channels may be formed by a
manufacturing process or by a biological developmental process that
may include at least one of vasculogenesis, angiogenesis, or
tubulogenesis. The one or more biological agents, the biological
agent gradient, the pressure, and/or the oxygen tension gradient
may further direct development, differentiation, and/or functioning
of the embryoid body or organoid. The wild-type population of cells
may comprise induced pluripotent stem cells (iPSCs) or
iPSCs-derived patent-specific cell lines.
In the above methods, the step of embedding the embryoid body or
organoid in the tissue construct comprises depositing one or more
cell-laden filaments each comprising a plurality of viable cells on
a substrate to form one or more tissue patterns, each of the tissue
patterns comprising one or more predetermined cell types,
depositing one or more sacrificial filaments on the substrate to
form a vascular pattern interpenetrating the one or more tissue
patterns, each of the sacrificial filaments comprising a fugitive
ink, depositing the embryoid body or organoid within the vascular
pattern, at least partially surrounding the one or more tissue
patterns and the vascular pattern with an extracellular matrix
composition, and removing the fugitive ink, thereby forming the
tissue construct comprising the embryoid body or organoid embedded
therein.
In certain embodiments, at least some portion of the one or more
cell-laden filaments may comprise the one or more biological
agents.
One or both of the first and second vascular networks comprise
microfluidic channels.
In certain further embodiments, a plurality of the embryoid bodies
or organoids may be embedded in the tissue construct. The embryoid
bodies or organoids may comprise different phenotypes or may
comprise the same phenotype.
In certain embodiments, the above methods, wherein an array of the
tissue constructs is present, wherein the embedding, exposing and
vascularizing may be carried out in each tissue construct.
Certain further embodiments relate to a functional human tissue or
an array of functional human tissues formed by the method described
herein.
These and other features and advantages of the invention will
become apparent upon consideration of the following detailed
description of the presently preferred embodiments, viewed in
conjunction with the appended drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
FIG. 1 depicts results of a study by Lancaster et al. (Lancaster et
al., 2013) where the cerebral organoids developed in vitro lacked a
perfusable vascular network.
FIG. 2 depicts an illustration of the concept of growing a
developing embryoid body or organoid with an internal developing
vascular plexus beginning with a single cell to vascularized
organ.
FIG. 3A depicts an illustration of a strategy to develop cell
organoids inside a 3D printed perfusion system. The `original
strategy` involves eliciting angiogenesis from endothelial cell
lined printed microchannels, whereby the sprouting endothelium
invades the implanted organoids.
FIG. 3B depicts an illustration of an alternative strategy to
develop cell organoids inside a 3D printed perfusion system. The
`revised strategy` involves eliciting angiogenesis from a
population of endothelial cells contained within the organoid or
embryoid body and attracting the sprouts towards the printed
channels, whereupon they connect the arterial and venous
networks.
FIG. 4 depicts a strategy for creating perfusable vascularized
organoids.
FIG. 5A depicts a photograph of a spinning bioreactor with cerebral
organoids.
FIG. 5B depicts an organoid grown in a spinning bioreactor at day
14.
FIG. 5C depicts an organoid grown in a spinning bioreactor at day
22.
FIG. 6 depicts a schematic illustration of the culture system used
to develop embryoid bodies or organoids from iPSCs and exemplary
images or organoids taken at each stage of embryoid body or
organoid development in vitro.
FIG. 7A shows an image of an organoid taken at T=0 d.
FIG. 7B shows an image of a developing organoid taken at T=day
1.
FIG. 7C shows an image of an organoid harvested at T=day 1.
FIG. 7D shows an image of a developing organoid taken at T=day
5.
FIG. 7E shows an image of a developing organoid taken at T=day
7.
FIG. 7F shows an image of a developing organoid taken at T=day
8.
FIG. 7G shows an image of a developing organoid taken at T=day
10.
FIG. 7H shows an image of a developing organoid taken at T=day 11
in matrigel (MG).
FIG. 7I shows an image of a developing organoid taken at T=day 12
in matrigel (MG).
FIG. 8A depicts a photograph of a cultured cerebral organoid with
internal plexus.
FIG. 8B depicts a photograph of a cultured cerebral organoid with
internal plexus.
FIG. 9 depicts a schematic illustration of a method of producing a
mixed population of wild-type and inducible cells.
FIG. 10 depicts a schematic illustration of a method of producing a
multi-population embryoid body or organoid.
FIG. 11 depicts a schematic illustration of a DNA delivery element
used for delivery of multiple transcription factors into cells to
produce mixed population organoids.
FIG. 12 depicts a schematic illustration of a strategy for creating
perfusable vascularized organoids.
FIG. 13 depicts vascular spheres derived from embryoid bodies
undergoing sprouting.
FIGS. 14A-14G depict a method of producing a network of vascular
channels in the extracellular matrix composition
FIG. 15 shows an exemplary design of a printed mold or an interface
structure.
FIG. 16 shows an exemplary 3D printing method of custom perfusion
chips.
FIG. 17 shows printing of vascular tissues.
FIG. 18 shows printing of vascularized tissues.
FIG. 19 depicts two, spanning, non-intersecting branched vascular
networks (i.e., artero-venous plexus) created to enable natural
capillary development to connect arterial and venous networks.
FIG. 20A depicts a top-view of printed artero-venous networks of
Pluronic F-127 sacrificial filaments that can be used to generate
an arterio-venous plexus after casting in a gel, cooling and
removing the liquefied sacrificial Pluronic F-127.
FIG. 20B shows the Pluronic-F127 structure of FIG. 20A printed
inside a printed silicone perfusion chip.
FIG. 20C shows how luer connectors can connect to the silicone chip
to enable connection to an external pump.
FIG. 20D shows how luer connectors can connect to the silicone chip
to enable connection to an external pump.
FIG. 21 depicts a schematic illustration of introducing the
embryoid body into the 3D printed vascular network.
FIG. 22A shows embedding embryoid bodies into vascularized tissues
(day 2).
FIG. 22B shows embedding embryoid bodies into vascularized tissues
(day 3).
FIG. 22C shows embedding embryoid bodies into vascularized tissues
(day 5).
FIG. 22D shows an exemplary perfusion chip that is connected to an
external for implanting and perfusing the sprouting organoid.
FIG. 23 depicts a strategy for promoting organoid and vascular
development and delivery of factors.
FIG. 24 shows endothelial vascular channels created by the
described method. HUVECs are in evacuated GelMA gel.
FIG. 25 shows a photograph of vascularized tissues.
FIG. 26 shows a process of active perfusion of vascularized
tissues.
FIG. 27, left image, shows organoids analyzed by immunofluorescence
for nestin (green) (a neural progenitor marker, and an endothelial
marker), and Sox 1 (a neural progenitor marker) (red); FIG. 27,
right image, shows the tubular morphology of the cells.
FIG. 28 depicts organoids stained for presence of neuronal
structures.
FIG. 29 depicts vascularized cerebral organoids from iPSCs.
FIG. 30 shows cerebral organoids within perfusable vascularized
matrices.
FIG. 31 shows `common sense` approach of mixing human
umbilical-vein endothelial cells (HUVECs) with induced pluripotent
stem cells (iPSCs).
FIG. 32 depicts organoids produced by combining neuronal and
endothelial protocols into a hybrid protocol.
FIG. 33 shows that populations of iPSCs that have been transformed
with a doxycycline inducible promoter for a different transcription
factors can be directly-differentiated to endothelial cells with
varying degrees of efficiency when doxycycline is added to mTeSR1
medium.
FIG. 34 shows flow cytometry data for two endothelial genes,
PECAM-1 (also known as CD31) and vascular endothelial cadherin
(VECad).
FIG. 35 shows iPSCs that were transformed with a dox-inducible ETV2
vector and cultured in mTeSR1 containing dox for 5 days.
FIG. 36 shows a time series of phase contrast micrographs of an
dox-inducible ETV2 embryoid body harvested from Aggrewells.TM. at
day 3 and cultured in a droplet of Matrigel bathed in neural
induction medium containing doxycycline.
FIG. 37 depicts vascular sprouting in 10 days, cultured in matrigel
using cerebral organoid culture conditions with and without
doxycycline, VEGF or PMA.
FIG. 38 shows embryoid bodies formed using a suspension of
doxycycline inducible ETV2 expressing iPSCs prepared in Matrigel as
described in Example 10.
FIG. 39 shows embryoid bodies formed using a suspension of
doxycycline inducible ETV2 expressing iPSCs.
FIG. 40 shows an immunofluorescence stained vascularized cerebral
organoid.
FIG. 41 depicts a 3D printing apparatus (left) used to create an
organoid with vascular sprouts (right).
DETAILED DESCRIPTION
Embryoid bodies or organoids (e.g., cerebral organoids) are a
promising platform for studying tissue development processes (e.g.,
neurodevelopment processes) in a three dimensional, biomimetic
environment. However, previously developed organoids lacked a
perfusable vasculature. Due to a lack of a pervasive, perfusable
vasculature, the organoids were developing necrotic cores once
their size exceeded approximately 1 mm in diameter (FIG. 1;
Lancaster et al., 2013)).
A new approach has been developed and described in the present
disclosure for creating vascularized embryoid bodies or organoids
via three-dimensional (3D) bioprinting. This highly scalable
platform enables the fabrication of engineered embryoid bodies or
organoids in which vasculature, multiple cell types and optionally
other functional chemical substances, such as drugs, toxins,
proteins and/or hormones, are programmably placed at desired
locations within an extracellular matrix. This technique may lead
to the rapid manufacturing of functional 3D tissues (i.e., "tissue
constructs") and organs needed for studies of tissue development
and disease, as well as transplantation. The inventive vascularized
embryoid bodies or organoids can also be used as a research tool to
study the effects of any external (e.g. drugs or other stimuli) or
internal (mutations) influences on growth and activity of cells in
the tissue.
Examples of organ, embryoid body, organoid, or tissue constructs
that can be produced by the described methods include, but are not
limited to, thyroid, pancreas, ureters, bladder, urethra, adrenal
glands, lung, liver, pineal gland, pituitary gland, parathyroid
glands, thymus gland, adrenal glands, appendix, gallbladder,
spleen, prostate gland, reproductive organs, neural and vascular
tissue.
As such, certain embodiments relate to methods of creating
vascularized developing embryoid bodies or organoids (e.g.,
cerebral organoids) to enable nutrient delivery via perfusion
necessary for generation of larger, more complex embryoid bodies or
organoids for transplantation and drug screening applications, as
well as for fundamental, long term studies of organogenesis. A
printed vascularized embryoid body or organoid and a method of
creating the embryoid body or organoid with an internal developing
vascular plexus are described herein. FIG. 2 provides an
illustration of the concept of growing a developing embryoid body
or organoid with an internal developing vascular plexus adjacent to
brain microvascular endothelial cell (BMEC)-lined printed
microchannels beginning with a single cell to a vascularized organ
that has anastomosed with the adjacent channels. The process
combines a 3D printing approach with developmental biology to
generate vascularized, functional human tissues for drug
development and regenerative medicine applications. Previous
technologies described approaches of developing avascular organoids
or printing blood vessels to maintain adult cell viability. In
contrast, described here are methods that employ combination of
autologous, printed blood vessels with developing embryoid bodies
or cell organoids inside a 3D printed perfusion system.
As used herein and in the appended claims, the singular forms "a,"
"and," and "the" include plural referents unless the context
clearly dictates otherwise. Thus, for example, reference to "a
protein" includes a plurality of such proteins and reference to
"the progenitor cell" includes reference to one or more progenitor
cells known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods, compositions,
devices and materials are described herein.
The term "embryoid body" refers to a plurality of cells containing
pluripotent or multipotent stem cells formed into a three
dimensional sphere, spheroid, or other three dimensional shape.
The term "organoid" refers to an embryoid body whose cells have
undergone a degree of differentiation.
The embryoid body or organoid is created by culturing at least one
of pluripotent stem cells, multipotent stem cells, progenitor
cells, terminally differentiated cells, endothelial cells,
endothelial progenitor cells, immortalized cell lines, or primary
cells, which will be described in detail below.
We acknowledge that the distinction between an embryoid body and
organoid remains undefined, and the use of the terms should be
considered interchangeable.
The embryoid body or organoid may be a cerebral organoid, thyroid
organoid, intestinal organoid, gut organoid, hepatic organoid,
pancreatic organoid, gastric organoid, kidney organoid, retinal
organoid, cardiac organoid, bone organoid, and epithelial
organoid.
The term "cerebral organoid" refers to an artificial
three-dimensional tissue culture created by culturing human
pluripotent stem cells in, e.g., a three-dimensional rotational
bioreactor. Cerebral organoids may be synthesized tissues that
contain several types of nerve cells and have anatomical features
that resemble mammalian brains. For example, cerebral organoids can
comprise a heterogenous population of cells of at least two
different progenitor and neuronal differentiation layers. The
cerebral organoids can display heterogeneous regionalization of
various brain regions as well as development of complex,
well-organized cerebral cortex. Cerebral organoids are most similar
to layers of neurons called the cortex and choroid plexus. In some
cases, structures similar to the retina, meninges and hippocampus
can form as well.
The term "internal plexus" refers to an interconnected network of
vascular endothelial cells that resides inside of, and/or on the
surface of a developing embryoid body or organoid.
A pattern or network that "interpenetrates" another pattern or
network in a printed tissue construct may be understood to comprise
one or more filaments, channels or portions that are layered with,
partially or completely overlapping, partially or completely
underlapping, surrounding, embedded within, and/or interwoven with
one or more filaments, channels or portions of the other pattern or
network. A filament "deposited on a substrate" may be understood to
be deposited directly on the substrate or directly on another
filament, channel or portion previously deposited or formed on the
substrate.
The term "PKC" is used as an acronym for protein kinase C, which
may refer to any of the isoforms of protein kinase C, including
PKC-alpha, PKC-beta1, PKC-beta2, PKC-gamma, PKC-delta, PKC-epsilon,
PKC-eta, PKC-theta, PKC-iota, and PKC-zeta.
The term "orthogonal promoters," refers to two different promoter
designs for which there are independent cues for gene induction.
For example, if there are two inducible genes, `gene 1` activated
by `promoter 1`, and `gene 2` activated by `promoter 2`, then
promoter 1 and 2 are orthogonal if both of the following statements
hold true:
1) There exists a `cue 1` that specifically induces the expression
of `gene 1` without directly affecting the expression of `gene 2`;
and
2) There exists a `cue 2` that specifically induces the expression
of `gene 2` without directly affecting the expression of `gene
1`.
The term "sprouts," or more specifically, "endothelial sprouts,"
refers to endothelial structures that have either undergone
angiogenesis or vasculogenesis to generate tubular structures.
The term "functional" as it refers to generating human tissue,
means that the tissue synthesized according to the described
methods have the same or similar functions to the organ intended to
be created.
As shown in FIGS. 3A-B, two possible strategies to develop cell
organoids inside a 3D printed perfusion system are considered.
In the first strategy, shown schematically in FIG. 3A, stem cells
are cultured in embryoid body growth medium, for example
Aggrewell.TM. medium from StemCell Technologies Inc. (AW) to
develop an embryoid body and, separately, in Brain Microvasculature
Differentiation Medium (BMEC) (Lippmann E S, et al. Derivation of
blood-brain barrier endothelial cells from human pluripotent stem
cells. Nat Biotechnol 30(8):783-91 (2012)) to develop brain
microvascular endothelial cells. The BMECs are then introduced into
3D printed, perfusable arterial microchannels and are allowed to
adhere to the channel walls. Next, a gradient of biological factors
may added to the venous channel to induce sprouting angiogenesis
that anastomoses with an internal vascular plexus in the developing
organoid. Once the endothelial sprouts span the space between the
arterial and venous network, the embryoid body or organoid can be
directly perfused via an external pump.
In the second strategy, shown schematically in FIG. 3B, stem cells
can be incubated in AW to produce an embryoid body, which is then
incubated in BMEC or neural induction medium (NIM) (Lancaster et
al., 2013) to produce a sprouting embryoid body. The sprouting
embryoid body is then incubated in NIM with EGM and placed into a
3D printed perfusion system to produce a vascularized organ.
The second strategy is further illustrated in FIG. 4, wherein the
cerebral organoids grown in vitro are placed within printed
vasculature, embedded and grown on a chip.
Methods for culturing and differentiating stem cells into neuronal
cells and tissues are known from Eiraku (2008), US 2011/0091869 A1
and WO 2011/055855 A1, contents of which are incorporated by
reference in their entirety. Methods described in U.S. Pat. Pub.
No. US 2015/0330970 to Lancaster et al. and Lancaster et al.,
"Cerebral organoids model human brain development and
microcephaly," Nature 501, 373-379 (2013)), incorporated by
reference herein, can be used in the first step of obtaining the
embryoid bodies or organoids, especially the steps of providing a
multicellular aggregation of pluripotent stem cells and culturing
the multicellular aggregation in neural induction medium.
The cells used to produce embryoid bodies or organoids (including
all further embodiments related thereto), are human cells or
non-human primate cells, pluripotent stem cells, multipotent stem
cells, progenitor cells, terminally differentiated cells,
endothelial cells, endothelial progenitor cells, immortalized cell
lines, or primary cells.
A "pluripotent" stem cell is not able to grow into an entire
organism, but is capable of giving rise to cell types originating
from all three germ layers, i.e., mesoderm, endoderm, and ectoderm,
and may be capable of giving rise to all cell types of an organism.
Pluripotency can be a feature of the cell per se, e.g. in certain
stem cells, or it can be induced artificially. E.g. in certain
embodiments, the pluripotent stem cell is derived from a somatic,
multipotent, unipotent or progenitor cell, wherein pluripotency is
induced, Such a cell is referred to as "induced pluripotent stem
cell" or "iPSC" herein. The somatic, multipotent, unipotent or
progenitor cell can, e.g., be used from a patient, which is turned
into a pluripotent cell, that is subject to the described methods.
Such a cell or the resulting tissue culture can be studied for
abnormalities, e.g. during tissue culture development according to
the described methods. A patient may, e.g., suffer from a
neurological disorder or cerebral tissue deformity. Characteristics
of the disorder or deformity can be reproduced in the described
embryoid bodies or organoids and investigated.
A "multipotent" cell is capable of giving rise to at least one cell
type from each of two or more different organs or tissues of an
organism, wherein the cell types may originate from the same or
from different germ layers, but is not capable of giving rise to
all cell types of an organism.
In contrast, a "unipotent" cell is capable of differentiating to
cells of only one cell lineage.
A "progenitor cell" is a cell that, like a stem cell, has the
ability to differentiate into a specific type of cell, with limited
options to differentiate, with usually only one target cell. A
progenitor cell is usually a unipotent cell, it may also be a
multipotent cell, and often has a more limited proliferation
capacity.
Preferably, the described embryoid body or organoid is created by
culturing initial populations of pluripotent or multipotent stem
cells.
In certain embodiments, the embryoid bodies or organoids can be
obtained from culturing pluripotent stem cells. In principle, the
cells may also be totipotent, if ethical reasons allow. A
"totipotent" cell can differentiate into any cell type in the body,
including the germ line following exposure to stimuli like that
normally occurring in development. Accordingly, a totipotent cell
may be defined as a cell being capable of growing, i.e. developing,
into an entire organism.
The cells used in the methods according to the present invention
are preferably not totipotent, but (strictly) pluripotent.
The culturing methods are known in the art. For example, culturing
can take place on a low-adhesion substrate (Doetschman T C, et al.,
The in vitro development of blastocyst-derived embryonic stem cell
lines: formation of visceral yolk sac, blood islands and
myocardium. J Embryol Exp Morphol 87:27-45 (1985)), via a hanging
drop method (Reubinoff B E, et al., Embryonic stem cell lines from
human blastocysts: somatic differentiation in vitro. Nat Biotechnol
18(4):399-404 (2002)), via aggregation in microwells (Mohr J C, et
al., 3-D microwell culture of human embryonic stem cells.
Biomaterials 27(36):6032-42 (2006), via aggregation in
microchannels (Onoe H, et al., Differentiation Induction of Mouse
Neural Stem Cells in Hydrogel Tubular Microenvironments with
Controlled Tube Dimensions. Adv Healthc Mater. (2016),
doi:10.1002/adhm.201500903), or by using a spinning bioreactor
(Carpenedo R L, et al., Rotary suspension culture enhances the
efficiency, yield, and homogeneity of embryoid body
differentiation. Stem Cells 25(9):2224-34 (2007)) (FIG. 5A). FIGS.
5B and 5C show organoids grown a spinning bioreactor at day 14 and
day 22, respectively.
A typical embryoid body or organoid protocol, according to the
described methods starts with isolated embryonic or pluripotent
stem cells (e.g., induced pluripotent stem cells, or iPS cells, or
iPSCs).
The organoid culture is in vitro grown (culturing step), i.e., it
is not an isolated organ, such as brain or Kidney from an animal
during any stages. Since it is grown from human pluripotent stem
cells, this allows growth of human tissue without the need to
obtain human fetal tissue samples.
For example, during the step of culturing the aggregate, the
pluripotent stem cells can be induced to differentiate into a
tissue (e.g., neural tissue) containing at least one of pluripotent
stem cells, multipotent stem cells, progenitor cells, terminally
differentiated cells, endothelial cells, endothelial progenitor
cells, immortalized cell lines, or primary cells. For providing a
multicellular aggregation, it is, e.g., possible to culture
pluripotent stem cells from the multicellular aggregates. For
example, FIG. 6 shows a schematic illustration of the culture
system used to develop embryoid bodies or organoids from iPSCs and
exemplary images or organoids taken at each stage of embryoid body
or organoid development in vitro. Differentiation of embryoid
bodies towards early cerebral organoids can be seen by the
development of neuroepithelial rosettes by day 8.
FIGS. 7A-I shows images of various organoids taken at various times
during development and in various media. This will be described in
detail below in the Examples section.
Exemplary media for culturing embryoid bodies or organoids include,
but are not limited to, Aggrewell.TM. medium (AW) commercially
available from StemCell Technologies, Inc., neural induction medium
(NIM) comprising DMEM/F12 medium, supplemented with 1:100 N2
supplement, 1 .mu.g/ml heparin sulfate, 1 mM glutamax, and MEM
non-essential amino acids. The small molecule smad inhibitor
SB431542 can be added to NIM at 10 nM concentration, and the
protein noggin can be added to enhance neural specification.
Cerebral organoids can be further differentiated in neural
differentiation medium, phase 1 (NDM1) comprising a 1:1 mix of
DMEM/F12 and Neurobasal medium supplemented with 1:200 N2
supplement, 1:100 B27 supplement without vitamin A, 3.5 .mu.L/L of
2-mercaptoethanol, 1:4000 insulin, 1:100 glutamax, and 1:200
MEM-non essential amino acids. Further cerebral maturation may be
achieved by culturing the organoids in Neural differentiation
medium, phase 2 (NDM2) including, e.g., a 1:1 mix of DMEM/F12 and
Neurobasal medium supplemented with 1:200 N2 supplement, 1:100 B27
supplement with vitamin A, 3.5 .mu.L/L of 2-mercaptoethanol, 1:4000
insulin, 1:100 glutamax, and 1:200 MEM-non essential amino
acids.
In certain embodiments, endothelial cells may be encouraged to
undergo proliferation and specification to a brain microvascular
phenotype by culturing the cells in brain microvascular endothelial
cell (BMEC) medium including, e.g., endothelial serum-free medium
supplemented with 20 ng/ml of FGF, 1% platelet-poor plasma-derived
bovine serum, and 10 .mu.M retinoic acid.
In certain embodiments, various biological agents or factors may be
used in combination with the media. Exemplary biological agents or
factors that may be used in the described method include, e.g.,
basic FGF, noggin, the small molecule TGF-beta inhibitor SB431542,
Activin A, BMP-4, Wnt, epidermal growth factor (EGF), ascorbic
acid, retinoic acid, bovine brain extract, heparin, hydrocortisone,
gentamicin, fetal bovine serum, Insulin-like growth factor (IGF),
and vascular endothelial growth factor (VEGF).
In certain embodiments, relating to synthesizing cerebral
organoids, during the development, the cell aggregates can form
polarized neuroepithelial structures and a neuroepithelial sheet,
which will develop several round clusters (rosettes). These steps
can be controlled by neural induction medium as described by Eiraku
(2008), US 2011/0091869 A1 and WO 2011/055855 A1.
In the absence of neural induction medium, e.g., by using standard
differentiation media, the method may include culturing in a three
dimensional matrix, preferably a gel, especially a rigid stable
gel. As such, in certain embodiments, the method also includes a
step of culturing the cell aggregates in a three dimensional
matrix, preferably a gel, which can result enhanced epithelial
polarization and improved cortical layer formation. For example,
further expansion of neuroepithelium and/or differentiation can be
observed with embryoid bodies or organoids cultured in a three
dimensional matrix.
A suitable three dimensional matrix may comprise collagen type 1 or
matrigel. In certain embodiments, the three dimensional matrix
comprises extracellular matrix from the Engelbreth-Holm-Swarm tumor
or any component thereof such as laminin, collagen, preferably type
4 collagen, entactin, and optionally further heparan-sulfated
proteoglycan or any combination thereof. Such a matrix is Matrigel.
Matrigel was previously described in U.S. Pat. No. 4,829,000, which
is incorporated by reference in its entirety.
In certain embodiments, the matrix comprises a concentration of at
least 3.7 mg/ml containing in parts by weight about 60-85% laminin,
5-30% collagen IV, optionally 1-10% nidogen, optionally 1-10%
heparan sulfate proteoglycan and 1-10% entactin. Matrigel's solid
components usually comprise approximately 60% laminin, 30% collagen
IV, and 8% entactin. Entactin is a bridging molecule that interacts
with laminin and collagen. The three dimensional matrix may further
comprise growth factors, such as any one of EGF (epidermal growth
factor), FGF (fibroblast growth factor), NGF, PDGF, IGF
(insulin-like growth factor), especially IGF-1, TGF-.beta., tissue
plasminogen activator. The three dimensional matrix may also be
free of any of these growth factors.
In certain embodiments, the three dimensional matrix may be a three
dimensional structure of a biocompatible matrix. It may include
collagen, gelatin, chitosan, hyaluronan, methylcellulose, laminin
and/or alginate. The matrix may be a gel, in particular a hydrogel.
Organo-chemical hydrogels may comprise polyvinyl alcohol, sodium
polyacrylate, acrylate polymers and copolymers with an abundance of
hydrophilic groups. Hydrogels comprise a network of polymer chains
that are hydrophilic, sometimes found as a colloidal gel in which
water is the dispersion medium. Hydrogels are highly absorbent
(they can contain over 99% water) natural or synthetic polymers.
Hydrogels also possess a degree of flexibility very similar to
natural tissue, due to their significant water content.
After the expansion, the cell aggregates can be cultured in
suspension culture, preferably a bioreactor, such as a spinning
bioreactor (FIG. 5A). The culturing in suspension culture is
preferably also in the absence of neural induction medium. A
suitable medium is a standard differentiation medium. "A spinning
bioreactor" refers to a device or system meant to grow cells or
tissues in the context of cell culture, as shown in FIG. 5A. These
devices are being developed for use in tissue engineering or
biochemical engineering. For example, a suitable spinning
bioreactor can be purchased from Wheaton Inc., or a Rotary Cell
Culture System can be purchased from Synthecon.
The cells in the bioreactors may be cultured in a proteinaceous
matrix (such as Matrigel) that supports three-dimensional growth.
FIGS. 5B and 5C show cerebral organoids at day 14 and day 22,
respectively, developing in the spinning bioreactor.
FIGS. 7A-7I also show cerebral organoids developing in spinning
bioreactors, scale bars=100 .mu.m.
As shown in FIGS. 8A-8B, organoids cultured according to the
described methods form an internal vascular plexus as visualized by
a network of endothelial cells identified by an antibody stain for
PECAM-1 (CD-31). The specific media conditions to produce the
organoid of FIG. 8A include Aggrewell.TM. medium (3 days), neural
induction medium (5 days), neural differentiation medium (phase 1;
5 days) and neural differentiation medium (phase 2; 5 days). The
specific media conditions to produce the organoid of FIG. 8B
include Aggrewell.TM. medium (3 days), brain microvascular
endothelium medium (5 days), neural differentiation medium (phase
1; 5 days) and neural differentiation medium (phase 2; 5 days).
After a set period of time the organoids grow mature enough for
study, or for implanting into a scaffold or biscaffold replete with
3D printed vasculature.
Importantly, prior to, during and/or after the implanting or
embedding, the embryoid body or organoid is further differentiated
using a combination of NIM, NDM1, EGM-2 or NDM2 media into a tissue
containing at least one of pluripotent stem cells, multipotent stem
cells, progenitor cells, terminally differentiated cells,
endothelial cells, endothelial progenitor cells, immortalized cell
lines, or primary cells.
In the embodiments described above, stem cells (e.g., iPSCs) are
cultured to form a cell aggregate, the pluripotent stem cells can
be induced to differentiate into a tissue (e.g., neural tissue)
containing at least one of pluripotent stem cells, multipotent stem
cells, progenitor cells, terminally differentiated cells,
endothelial cells, endothelial progenitor cells, immortalized cell
lines, or primary cells. For providing a multicellular aggregation,
it is, e.g., possible to culture pluripotent stem cells from the
multicellular aggregates. For example, FIG. 6 shows a schematic of
the culture system used to develop organoids from iPSCs and
exemplary images or organoids taken at each stage.
In certain alternative embodiments, embryoid bodies or organoids
comprising at least two different populations of organoid or
embryoid body cells (i.e., a first population of embryoid body or
organoid cells and a second population of the embryoid body or
organoid cells) can be produced and later vascularized. For
example, in certain embodiments, the embryoid body or organoid can
comprise multiple populations of cells (i.e., at least two
different cell lineages; FIG. 9), such as endothelial and neuronal,
obtained by differentiation of iPSCs using the same culture
condition.
As shown schematically in FIG. 10, the method of producing the
multi-population embryoid body or organoid includes culturing a
wild-type population of cells and a genetically-engineered
inducible population of cells in a medium, inducing direct
differentiation and/or transdifferentiation of the
genetically-engineered inducible population of cells into the first
population of the embryoid body or organoid cells, inducing
differentiation of the wild-type population of cells into the
second population of the embryoid body or organoid cells, and
thereby forming the embryoid body or organoid comprising at least
the first population of the embryoid body or organoid cells and the
second population of the embryoid body or organoid cells.
The terms "direct differentiation" or "directed differentiation"
refer to the culture of pluripotent or multipotent stem cells in a
condition that preferentially encourages the differentiation of the
stem cell to a specific, more differentiated state. For example, a
pluripotent stem cell may be cultured in a condition that results
in an enriched population of specific multipotent stem cells such
as neural progenitor cells. Alternatively, a multipotent stem cell
such as a neural stem cell may be directly differentiated into a
more differentiated state such as a neuron, astrocyte or
oligodendrocyte.
The term "transdifferentiation" refers to the conversion of one
cell type that may be a multipotent or unipotent stem cell, or a
terminally differentiated mature cell phenotype to a different cell
type that may be a different multipotent or unipotent stem cell, or
a terminally differentiated mature cell phenotype. For example, a
neural stem cell, a radial glia, or a neuron may be
transdifferentiated into an endothelial cell.
The genetically-engineered inducible population of cells may be
created by introducing a DNA delivery element (as illustrated in
FIG. 11) comprising at least one of constitutive promoter, small
molecule inducible promoter, cell-autonomous promoter, cell
non-autonomous promoter, selection marker, or a combination
thereof.
Examples of constitutive promoters include, e.g., EF1alpha, PGK,
Ubiquitin, and CMV. Examples of small molecule inducible promoters
include, e.g. doxycycline or cumate inducible promoters. Examples
of cell-autonomous promoters include, e.g., cell type-specific
promoters, such as DCX. Examples of cell non-autonomous promoter
include, e.g., heat induced and light induced promoters.
DNA delivery elements can be selected from lentiviral inverted
repeats, packaging signal (e.g., pLIX403 vector), transposon
integration elements (e.g., PiggyBac vector), episomal replication
elements. Alternatively, transient expression by electroporation or
lipofection can be used.
Selection markers may be selected from, e.g., drug resistance
markers (e.g. puromycin, neomycin, and blasticidin). Alternatively,
transient expression followed by dilution from cell division rather
than selection markers may be used.
Examples of specific transcription factors that may be used to
induce endothelial cells within any organoid (e.g. for vasculature)
and to produce mixed populations within organoids include
ETV2/ER71, FL11, ERG (Ginsberg et al. 2012 Cell), which induce
differentiation of mature amniotic cells to endothelial cells;
Gata2, FOXC1, FOXC2, HEY1, HEY2, SOX7, SOX18, PROX1 (Park et al.
2013 Circulation Research), which induce differentiation of stem
cells into various subtypes of endothelial cells (e.g. venous,
arterial, lymphatic); Brachyury/T, which may be used for possible
mesoderm induction, required for primitive streak formation in
vivo.
Examples of specific transcription factors that may be used to
induce neurons within any organoid (e.g. autonomic nervous system
control of internal organs) include NEUROG1/2 (Busskamp, et al.,
Molecular Systems Biology (2014)), which induce formation of
excitatory neurons; ASCL1 (Chanda, et al., Stem Cell Reports
(2014)), which induce formation of excitatory neurons; ASCL1, BRN2,
MYT1L, LHX3, HB9, ISL1, NGN2 (Son et al. 2011 Cell Stem Cell),
which induce formation of motor neurons; and ASCL1, MYT1L, KLF7
(Wainger, et al., Nature Neuroscience (2014)), which induce
formation of pain receptor neurons.
The first population of the embryoid body or organoid cells can
comprise pluripotent stem cells, multipotent stem cells, progenitor
cells, terminally differentiated cells, endothelial cells,
endothelial progenitor cells, immortalized cell lines, or primary
cells.
The second population of the embryoid body or organoid cells
comprises neural progenitor cells. The neural progenitor cells can
form at least one of excitatory neurons, inhibitory interneurons,
motor neurons, dopaminergic neurons, pain receptor neurons,
astrocytes, oligodendrocyte progenitor cells, oligodendrocytes.
The step of inducing direct differentiation and/or
transdifferentiation of the genetically-engineered inducible
population of cells can comprise introducing at least one cue
selected from the group consisting of transcription factors, drugs,
small molecules, growth factors, morphogens, hormones, DNA, shRNA,
siRNA, nanoparticles, mRNA, modified mRNA, heat, light, and
mechanical stimulation.
In certain embodiments, the direct differentiation may be
accompanied by a secondary induction of a different gene, e.g., a
second orthogonal induction. This secondary induction may occur at
an earlier time, simultaneously, or at a later time than the first
gene induction. The secondary gene induction may be via providing
at least one cue selected from the group consisting of
transcription factors, drugs, small molecules, growth factors,
morphogens, hormones, DNA, shRNA, siRNA, nanoparticles, mRNA,
modified mRNA, heat, light, and mechanical stimulation.
In certain embodiments, the cue selected for the secondary gene
induction is the same as the cue selected for the step of inducing
direct differentiation and/or transdifferentiation of the
genetically-engineered inducible population of cells.
Alternatively, the cue selected for the secondary gene induction is
different, and orthogonal, from the cue selected for the step of
inducing direct differentiation and/or transdifferentiation of the
genetically-engineered inducible population of cells.
In certain embodiment, the first population of the embryoid body or
organoid cells can undergo a further development due to induction
of a secondary gene. The induction of the secondary gene induces an
expression of a constitutively-active PKC protein thereby enhancing
at least one of a sprouting behavior of the first population of the
embryoid body or organoid cells, or neurite outgrowth. The first
population of the embryoid body or organoid cells may be
endothelial cells.
The ratio of the first population the embryoid body or organoid
cells to the second population of the embryoid body or organoid
cells may be 1:1, 1:2, 1:3. 1:4, 1:5, etc. or 5:1. 4:1. 3:1,
2:1.
The step of culturing may be in a differentiation medium. In
certain embodiments, the differentiation medium includes
doxycycline (DOX) or another drug.
Importantly, the concept of inducing not only differentiation, but
specific programmed cell behaviors by either adding a second gene
that is induced by the same signal, or adding a second orthogonal
induction cue like a different drug than doxycycline is described.
The application would be to induce the expression of a
constitutively-active PKC protein that dramatically enhances
sprouting behavior of endothelial cells. PKC also encourages
enhanced neurite outgrowth. It is important that the endogenous PKC
signaling that directs neural outgrowth in cerebral organoids is
not affected. Thus, by activating PKC in only the subset, sprouting
in the endothelial cells is specifically achieved.
Once, or before, the embryoid bodies or organoids grows to a size
at which it becomes oxygen or nutrient limited--typically once it
reaches approximately 1 mm in diameter, (1-22 days) and having the
desired characteristics described above, the embryoid body or
organoid is implanted or embedded into a scaffold or biscaffold
replete with 3D printed vasculature.
In certain embodiments, as shown in FIG. 13, embedding embryoid
bodies or organoids in, e.g., collagen (e.g., collagen I)
encourages vascular sprouting (from vascular spheres).
FIG. 12 schematically shows steps where the embryoid body is
embedded in a matrigel or collagen and then embedded into
vascularized matrix on a perfusable chip for arterial and venous
circulation.
Vascularized tissue constructs and methods of producing vascular
channels in the extracellular matrix composition using 3D printed
technology was previously described in WO2015/069619, which is
incorporated herein by reference in its entirety.
For example, vascular channels may be created by depositing one or
more sacrificial filaments, each comprising a fugitive ink and/or a
plurality of viable cells on the substrate to form a vascular
pattern. The vascular pattern can be partially or fully surrounded
by an extracellular matrix composition. The fugitive ink is then
removed to create a network of vascular channels in the
extracellular matrix composition (FIGS. 14A-G).
Advantageously, the composition may be designed to support the
attachment and proliferation of endothelial cells, which line
vascular channels providing a barrier to fluid diffusion, while
simultaneously facilitating homeostatic functions and helping
establish vascular niches specific to the various tissues. To
promote endothelialization, in some embodiments the sacrificial
filament(s) comprising the fugitive ink may further include a
plurality of endothelial cells or other viable cells. The cells may
be deposited along with the sacrificial filament and may remain in
the vascular channels after removal of the fugitive ink, as
illustrated in FIGS. 14A-14C. Direct cellularization of the
channels can be achieved if the cells adsorb to the channel walls
after liquidation of the fugitive ink. This approach may allow one
to incorporate viable cells into highly tortuous networks or small
channels that may be difficult to infill using direct injection due
to an increased resistance to flow. An exemplary printed tissue
construct including a channel formed by evacuation of a fugitive
ink comprising endothelial cells and Pluronic F127 is shown in
FIGS. 14D-14G, and is was previously described in the WO
2015/069619.
Specifically, one or more sacrificial filaments comprising a
fugitive ink and or a plurality of endothelial cells or other
viable cells may be deposited on a substrate to form a vascular
pattern. The vascular pattern comprises a two- or three-dimensional
interconnected arrangement or network of the one or more
sacrificial filaments. Removal of the fugitive ink after partial or
complete encapsulation with the extracellular matrix composition
creates a perfusable network of vascular channels. Because the
sacrificial filaments may be deposited in a 3D printing process
that involves extrusion through a micronozzle, it may be
advantageous for the fugitive ink to: (1) exhibit shear thinning
behavior; (2) exhibit a defined yield stress T.sub.y; and/or (3)
have a shear elastic modulus G' and a shear viscous modulus G''
modulus where G'>G'' at room temperature.
The substrate for deposition typically comprises a material such as
glass or other ceramics, PDMS, acrylic, polyurethane, polystyrene
or other polymers. In some embodiments, the substrate may comprise
living tissue or dehydrated tissue, or one of the extracellular
matrix compositions described above. The substrate may be cleaned
and surface treated prior to printing. For example, glass
substrates may undergo a silane treatment to promote bonding of the
cell-laden filaments to the glass substrate. In some embodiments,
it is envisioned that the substrate may not be a solid-phase
material but may instead be in the liquid or gel phase and may have
carefully controlled rheological properties, as described, for
example, in W. Wu et al., Adv. Mater. 23 (2011) H178-H183, which is
hereby incorporated by reference. In the work of Wu et al., a
fugitive ink was printed directly into synthetic hydrogels to
create network structures. However, these synthetic materials do
not support cell attachment and proliferation, limiting their use
to non-biological applications. In the present disclosure, an
extracellular matrix composition that facilitates cell attachment,
migration, proliferation, and tissue-specific function while
maintaining the appropriate rheology for printing is described. The
sacrificial filaments are embedded in the extracellular matrix
composition during printing, and thus the at least partial
surrounding of the vascular patterns with the extracellular matrix
composition occurs during deposition of each of the sacrificial
filaments. This includes arbitrarily complex 3D structures that may
require support material during printing. When the forming and
embedding of the vascular patterns occurs simultaneously, the
substrate onto which deposition occurs may be considered to be the
container that holds the extracellular matrix composition or the
extracellular matrix composition itself.
To form the extracellular matrix composition, a microgel (e.g., a
poly(acrylic acid) (PAA) microgel) may be used as a rheological
modifier and blended with one or more extracellular matrix
materials, as set forth previously, such as gelatin methacrylate. A
semi-interpenetrating polymer network (semi-IPN) may be formed.
Microgels may be understood to comprise colloidal gel particles
that are composed of chemically cross-linked three-dimensional
polymer networks. Microgels may act as sterically stabilized
colloids with only a shell and no core. They can vary in
composition and may include PAA, polystyrenes, PEG, and/or other
biomaterials. It is contemplated that a natural extracellular
matrix or biomaterial may be converted into a microgel form to
impart the ideal rheology. Examples of suitable biomaterials
include hyaluron, collagen, alginate, fibrin, albumin, fibronectin,
elastin, or matrigel. Alternatively, synthetic materials such as
PEG, acrylates, urethanes, or silicones may be modified in a
similar manner.
Representative rheological measurements of ink and matrix rheology
that are appropriate for embedded printing were previously
described in WO 2015/069619. In one example, a high molecular
weight (>1.25 MDa) PAA microgel may be used as a rheological
modifier and blended with gelatin-methacrylate (GelMa) to create an
extracellular matrix composition that supports the creation of
complex 3D vascular networks, which, in certain embodiments may be
endothelialized as described in WO 2015/069619. The transparency of
the extracellular matrix composition may be altered by varying the
degree of substitution and mesh size.
The method may further include, prior to surrounding or
encapsulating the vascular patterns with the extracellular matrix
composition, depositing one or more structural filaments layer by
layer on the substrate in a predetermined pattern to form a mold.
The structural filaments may comprise one or more structural
materials selected from among the exemplary extracellular matrix
compositions or extracellular matrix materials provided above. The
mold may hold the extracellular matrix composition during the
encapsulation and may remain as part of the tissue construct, or it
may be removed after processing. The structural filaments may
define the perimeter of the tissue construct on the substrate and
all or at least a portion of the three-dimensional shape of the
tissue construct out of the XY plane.
The mold may also have other functionalities besides defining the
shape of the construct. For example, the mold may serve as an
interface for perfusion of channels in a printed tissue construct.
FIG. 15 shows an exemplary design of a printed mold or an interface
structure. The mold, which may also be referred to as an interface
structure, can hold vascularized tissue in place during rocking by
immobilizing the tissue construct between a base portion of the
mold, which may comprise PDMS, and an overlying cover, which may
comprise glass.
The mold designs of FIG. 15 enables active pump-based perfusion and
include flow channels that are in fluid communication with (e.g.,
contiguous with) the vascular channels. Conduits that serve as flow
channels may be partially or fully embedded in the mold itself and
hollow tubes (e.g., metal tubes) may be used to interface with the
vascular channels. The exemplary mold shown in FIG. 15 has a wall
with multiple buttresses that contain the flow channels, which
include hollow pins extending into the interior of the mold, where
the tissue construct is fabricated. The vascular channels of the
tissue construct may be contiguous with apertures of the hollow
pins to enable flow to be introduced into the vascular channels
from tubing connected to the flow channels, and fluid may be
removed from the vascular channels through one or more other
apertures.
In one example, the mold may be formed of an elastomeric silicone,
a structural material known to be viscoelastic, non-toxic,
biocompatible, and capable of forming reversible press-to-fit
seals. The structural material may be 3D printed to form one or
more uncured structural filaments comprising one or more of
silicone, epoxies, esters of acrylic acid, or one of the
extracellular matrix compositions provided above. After printing is
complete, the structural filament(s) may be cured (e.g. by heating
or photopolymerizing) for a suitable time duration (e.g., about one
hour or more), after which the mold may exhibit the desired
material properties.
The encapsulation of the vascular patterns may comprise casting a
liquified matrix precursor into the mold and gelling the matrix
precursor to form the extracellular matrix composition. Casting of
the matrix precursor may take place at a temperature of from about
25.degree. C. to about 40.degree. C. For example, gelatin
methacrylate, or GelMA, may be cast at a temperature of about
37.degree. C. After casting, the matrix precursor may be cooled
(e.g., to about 15.degree. C. in the case of GelMA) to form a rigid
physical gel. Alternatively, the encapsulation may occur during
deposition of the vascular patterns in an embedded or
omni-directional 3D printing process, as indicated above. It is
also contemplated that the extracellular matrix composition may be
deposited by filament deposition, similar to the sacrificial
filaments. For example, one or more ECM filaments comprising the
extracellular matrix composition may be extruded from a nozzle and
deposited on the substrate layer by layer to build up the desired
3D geometry. In such a case, it may not be necessary to employ a
mold to contain the extracellular matrix composition.
The extracellular matrix composition may be cured before or after
removal of the fugitive ink to form a permanently chemically
cross-linked structure. Depending on the extracellular matrix
composition, the curing may entail heating, UV radiation or
chemical additives (e.g., enzymatic curing).
Any or all of the filaments deposited on the substrate--including
the one or more sacrificial filaments defining the interpenetrating
vascular pattern or a functional channel pattern, the one or more
structural filaments that may define the mold, and/or the one or
more ECM filaments that may yield the extracellular matrix
composition--may be extruded from a nozzle before being deposited
on the substrate. The extrusion process was previously described in
WO 2015/069619, which is incorporated herein in its entirety.
The vascular network may be a two- or three-dimensional
interconnected arrangement of vascular channels. The network may
include one or more -furcations (e.g., bifurcations, trifurcations,
etc.) from a parent vascular channel to a plurality of branching
vascular channels. The network may have a hierarchical branching
structure, where larger diameter channels branch into smaller
diameter channels. Some or all of the vascular channels may follow
a curved path, and thus may be considered to be curvilinear. All of
the vascular channels in the network may have the same diameter, or
at least one, some, or all of the vascular channels may have a
different diameter. In some cases, one or more of the vascular
channels may have a nonuniform diameter along a length thereof.
FIG. 16 shows exemplary 3D printing method of custom perfusion
chips.
Printing of vascularized tissues is shown in FIG. 17. In the shown
embodiment, the PDMS border is printed first, followed by
depositing filament comprising fugitive ink to create vascular
channels. The next step includes depositing a first cell-laden ink
and a second cell-laden ink. The step may be repeated 2-4 times
followed by infilling with the ECM. Next, as shown in FIG. 18, the
matrix is cooled (about 4.degree. C.) to evacuate fugitive ink to
produce a vascular network. Human umbilical vascular endothelial
cells (HUVECs) may then be introduced to create blood vessels.
In certain embodiments, as shown in FIG. 19, two, spanning,
non-intersecting branched vascular networks (i.e., artero-venous
plexus) are created to enable natural capillary development to
connect arterial and venous networks.
FIGS. 20A-20D show examples of a 3D printed interface device for
perfusing organoid bodies or organoids. Specifically, FIG. 20A
shows a top-view of printed networks of Pluronic F-127 sacrificial
filaments that can be used to generate an arterio-venous plexus
after casting in a gel, cooling and removing the liquefied
sacrificial Pluronic F-127. In this architecture, there are two
independent networks that intertwine without contacting each other,
as illustrated in FIG. 19. In this manner, a space a can be filled
with two independent channel networks, an arterial, and a venous
plexus; FIG. 20B shows the Pluronic-F127 structure of FIG. 20A
printed inside a printed silicone perfusion chip. This chip
facilitates the casting of the gel that surrounds the sacrificial
filaments, the removal of the sacrificial material, and the
subsequent active perfusion of the two independent channel
networks; FIG. 20C and FIG. 20D show how luer connectors can
connect to the silicone chip to enable connection to an external
pump.
Once the embryoid body or organoid and the vascularized matrix
comprising two, spanning, non-intersecting branched vascular
networks (i.e., a first vascular network and a second vascular
network, each vascular network vascular network comprising one or
more interconnected vascular channels) are prepared as described in
detail above, an embryoid body or organoid is embedded or implanted
into the vascularized matrix (FIG. 21). The one or more
interconnected vascular channels are formed by a manufacturing
process or by a biological developmental process that may include
at least one of vasculogenesis, angiogenesis, or tubulogenesis, as
described above.
The embryoid body or organoid may be introduced by casting a gel
around a column of pluronic, removing the pluronic to generate a
microwell, and placement of an embryoid body or organoid into the
microwell. FIGS. 22A-D show embedding embryoid bodies into
vascularized tissues. Specifically, FIGS. 22A-C show the embryoid
bodies on day 2, day 3 and day 5, respectively, following the
implantation. The red cells are HUVECs that line the printed
vascular channels. Two independent vascular channels surround the
central embryoid body or organoid (green) to provide nutrients and
oxygen, and remove waste products. FIG. 22D shows an exemplary
perfusion chip that is connected to an external for implanting and
perfusing the sprouting organoid.
In certain embodiments, prior to embedding the embryoid body or
organoid in the vascularized matrix, the embryoid body or organoid
may be encapsulated in an extracellular matrix material, as
described above. Preferably, the extracellular matrix material may
comprise a gel. Additional examples of matrices that may be used
for encapsulating the embryoid body or organoid include, but are
not limited to, at least one of collagen I, fibrin, matrigel,
gelatin, gelatin methacrylate, laminin, carbopol, NIPAM, PEG,
PHEMA, silk, hyaluronic acid, or combinations thereof.
Alternatively, in certain other embodiments, the embryoid body or
organoid is not encapsulated in an extracellular matrix material
prior to embedding it in the vascularized matrix.
In certain embodiments, the embryoid body or organoid is embedded
in a vascularized matrix by depositing the embryoid body or
organoid embedded in matrigel or collagen into a vascularized
matrix on a perfusable chip. As described above, the perfusable
chip includes outlets to provide arterial and venous circulation. A
strategy for creating perfusable vascularized organoids is
schematically shown in FIG. 12.
The embryoid body or organoid is then exposed to one or more
biological agents or factors, a biological agent gradient, a
pressure, and/or an oxygen tension gradient, thereby inducing
angiogenesis of capillary vessels to and/or from the embryoid body
or organoid (FIG. 23). The supporting fibroblasts and organoids or
embryoid bodies are shown in green and can be seen as growing due
to the perfused nutrients through the surrounding channels; and the
HUVECs are shown in red.
For example, one or more biological agents, a biological agent
gradient, a pressure, and/or an oxygen tension gradient encourages
the vascular plexus internal to the embryoid body or organoid to
sprout away from the developing embryoid body or organoid by means
of growth factors introduced to the 3D printed embryoid body or
organoid. Also, the one or more biological agents, the biological
agent gradient, the pressure, and/or the oxygen tension gradient
further direct development, differentiation, and/or functioning of
the embryoid body or organoid.
In certain embodiments, growth factors and oxygen may be directly
supplied to grow embryoid bodies or organoids via perfusion using
the perfusable chip (FIGS. 20A-D). Some examples of growth factors
that encourage connection of vasculature include, but are not
limited to, vascular endothelial growth factor (VEGF), basic
fibroblast growth factor (bFGF), sphingosine-1-phosphate (S1P),
phorbol myristate acetate (PMA), hepatocyte growth factor (HGF),
monocyte chemotactic protein-1 (MCP-1), the angiopoietin ANG-1, the
angiopoietin ANG-2, transforming growth factor beta (TGF-.beta.),
epidermal growth factor (EGF), human growth factor, matrix
metalloproteinases (MMP's), and histamine.
In certain embodiments, the embryoid body or organoid is exposed to
the one or more biological agents and/or the biological agent
gradient due to diffusion of the one or more biological agents
within the vascularized matrix. Alternatively, the embryoid body or
organoid is exposed to the one or more biological agents and/or the
biological agent gradient by localized deposition of materials
loaded with the one or more biological agents within the tissue
construct. Alternatively, the embryoid body or organoid is exposed
to the one or more biological agents and/or the biological agent
gradient by localized de-novo production of growth factors by
localized protein translation. Alternatively, the embryoid body or
organoid is exposed to the one or more biological agents and/or the
biological agent gradient via perfusion of one or both of the first
and second vascular networks with the one or more biological
agents.
In certain embodiments, only one of the first and second vascular
networks is perfused with the one or more biological agents.
In certain other embodiments, both the first and second vascular
networks are perfused with the one or more biological agents,
wherein a biological agent concentration in the first vascular
network is different than a biological agent concentration in the
second vascular network.
In certain alternative embodiments, both the first and second
vascular networks are perfused with the one or more biological
agents, wherein a biological agent concentration in the first
vascular network is the same as a biological agent concentration in
the second vascular network.
The one or more biological agents can include, but are not limited
to, one or more of the following: growth factors, morphogens, small
molecules, drugs, hormones, DNA, shRNA, siRNA, nanoparticles, mRNA,
modified mRNA.
Also, the biological agents can include one or more of the
following growth factors: vascular endothelial growth factor
(VEGF), basic fibroblast growth factor (bFGF),
sphingosine-1-phosphate (S1P), phorbol myristate acetate (PMA),
hepatocyte growth factor (HGF), monocyte chemotactic protein-1
(MCP-1), the angiopoietin ANG-1, the angiopoietin ANG-2,
transforming growth factor beta (TGF-.beta.), epidermal growth
factor (EGF), human growth factor, matrix metalloproteinases
(MMP's), and histamine.
In certain embodiments, an oxygen partial pressure gradient is
introduced to one or both of the first and second vascular networks
during perfusion. The oxygen partial pressure gradient may be
formed by introducing deoxygenated media into one of the first and
second vascular networks, and by introducing oxygenated media into
the other of the first and second vascular networks. In certain
embodiments, the perfusion may be carried out at a flow rate of
from about 1 microliter per minute to about 1 liter per minute. In
certain embodiments, one or both of the first and second vascular
networks may be subjected to a transmural pressure during the
perfusion.
In certain embodiments, following the exposure of the embryoid body
or organoid to one or more biological agents, the biological agent
gradient, the pressure, and/or the oxygen tension gradient, the
capillary vessels connect the first vascular network to the second
vascular network, thereby creating a single vascular network and a
perfusable tissue structure. In certain embodiments, the single
vascular network comprises an interpenetrating vascular network
and/or a branched interpenetrating vascular network. Preferably,
the single vascular network comprises interconnected arterial and
venous channels.
In certain embodiments, the first vascular network and the second
vascular network are independently addressable. In certain other
embodiments, the first vascular network and the second vascular
network are not in contact with each other.
In certain embodiments, the first vascular network comprises an
arterial plexus and the second vascular network comprises a venous
plexus.
In certain further embodiments, embedding the embryoid body or
organoid in the vascularized matrix comprises depositing one or
more cell-laden filaments each comprising a plurality of viable
cells on a substrate to form one or more tissue patterns, each of
the tissue patterns comprising one or more predetermined cell
types, depositing one or more sacrificial filaments on the
substrate to form a vascular pattern interpenetrating the one or
more tissue patterns, each of the sacrificial filaments comprising
a fugitive ink, depositing the embryoid body or organoid within the
vascular pattern, at least partially surrounding the one or more
tissue patterns and the vascular pattern with an extracellular
matrix composition, and removing the fugitive ink, thereby forming
the tissue construct comprising the embryoid body or organoid
embedded therein. At least some portion of the one or more
cell-laden filaments may comprise the one or more biological
agents. One or both of the first and second vascular networks may
comprise microfluidic channels.
In certain embodiments, a plurality of the embryoid bodies or
organoids is embedded in the vascularized matrix. The embryoid
bodies or organoids may comprise different phenotypes or may
comprise the same phenotype.
In certain embodiments, embedding the embryoid body or organoid in
the vascularized matrix may also comprise embedding the embryoid
body or organoid in an array of the vascularized matrices, wherein
the embedding, exposing and vascularizing is carried out in each
vascularized matrix.
As described above, the vascular plexus internal to the embryoid
body or organoid is encouraged to sprout away from the developing
embryoid body or organoid by means of, e.g., growth factors
introduced to the 3D printed embryoid body or organoid. After the
sprouting plexus reaches the 3D printed vessels, the embryoid body
or organoid may be perfused by use of a peristaltic pump and
perfusion device as outlined in the International Publication No.
WO 2015/069619. FIGS. 20A-20D and 20E-20G show exemplary designs of
printed molds or interface structures. The exemplary mold shown in
FIG. 15 is designed for passive rocking perfusion. The mold, which
may also be referred to as an interface structure, can hold
vascularized tissue in place during rocking by immobilizing the
tissue construct between a base portion of the mold, which may
comprise PDMS, and an overlying cover, which may comprise
glass.
In certain embodiments, the mold designs enable active pump-based
perfusion of a tissue construct and include flow channels that are
in fluid communication with (e.g., contiguous with) the vascular
channels of the tissue construct. Conduits that serve as flow
channels may be partially or fully embedded in the mold itself and
hollow pins (e.g., metal pins) may be used to interface with the
vascular channels. The exemplary mold shown in FIG. 15 has a wall
with multiple buttresses that contain the flow channels, which
include hollow pins extending into the interior of the mold, where
the tissue construct is fabricated. The vascular channels of the
tissue construct may be contiguous with apertures of the hollow
pins to enable flow to be introduced into the vascular channels
from tubing connected to the flow channels, and fluid may be
removed from the vascular channels through one or more other
apertures.
In an additional aspect, the invention provides a method of
investigating a developmental neurological tissue effect, e.g. a
defect, in particular a developmental defect, comprising decreasing
or increasing the expression in a gene of interest in a cell at any
stage during the described method.
Certain further embodiments relate to a method of screening a
candidate therapeutic agent suitable for treating a developmental
neurological tissue defect of interest. According to this aspect, a
candidate therapeutic drug can be screened for having an effect on
any cell with a mutation, which can be introduced as described
above. It is of course also possible to use cells of patients with
a given mutation, inducing pluripotent stem cell status and
performing the inventive methods to induce tissue development as
described above.
Of course, it is also possible to screen candidate drugs, e.g.
candidate therapeutic drugs, to have any effect on normal tissue as
well, without a mutation, which leads to an aberrant development.
Thus in yet another aspect, the invention relates to a method of
testing a candidate drug for neurological effects, comprising
administering a candidate drug to an artificial culture and
determining an activity of interest of the cells of said culture
and comparing said activity to an activity of cells to the culture
without administering said candidate drug, wherein a differential
activity indicates a neurological effect. Any kind of activity of
the inventive cells or tissue, including metabolic turn-over or
neuronal signaling can be searched for in a candidate drug. In
essence, the inventive highly differentiated tissue can be used as
a model for cerebral behavior testing on any effects of any drug.
Such a method might also be used to test therapeutic drugs,
intended for treating any kind of diseases, for having side-effects
on nerves, in particular brain tissue, as can be observed in the
inventive tissue culture.
Certain further embodiments relate to methods to obtain neuronal
cells. In particular, the invention provides a method of obtaining
a differentiated neural cell comprising the step of providing an
artificial culture and isolating a differentiated neural cell of
interest, or comprising the step of generating an artificial tissue
culture according to the invention further comprising the step of
isolating a differentiated neural cell of interest. Such cells
isolated from the inventive culture or tissue have the benefit of
representing similar morphological properties as cells isolated
from cerebral tissue of an non-human animal, as mentioned above, or
a human.
Certain additional embodiments relate to a functional human tissue
or an array of functional human tissues formed by the described
method.
EXAMPLES
Example 1
Printing Vascularized Tissues
To create 3D printed vascularized tissues, the following method
(illustrated in FIGS. 17 and 18) was used:
Step 1: PDMS border was 3D printed using SE 1700 from Dow
chemicals.
Step 2: Fugitive ink Pluronic F-127 or gelatin was 3D printed to
create vascular channels.
Step 3: Cell laden ink #1 comprising cells mixed in 10% wt/v
gelatin-fibrinogen or gelatin methacrylate (GelMA) hydrogels
hydrogels containing a photoinitiator Irgacure-2959 at 0.3% wt/v
was 3D printed.
Step 4: Cell-laden ink #2 I comprising cells mixed 10% wt/v gelatin
methacrylate hydrogels containing a photoinitiator Irgacure-2959 at
0.3% wt/v was 3D printed.
Steps 1-4 above were repeated.
The resultant fugitive ink and cell-laden filaments were infilled
with extracellular matrix comprising gelatin methacrylate at 10%
wt/v containing 0.3% wt/v Irgacure 2959 photoinitiator.
The structure was then exposed to chemically crosslink the gelatin
methacrylate.
Conditions:
Green with blue nuclei: GFP HNDFs stained with DAPI
Red with blue nuclei: RFP HUVECs stained with DAPI
Non-fluorescent with blue nuclei: 10T1/2 MFs
The ECM filed construct was then cooled to 4.degree. C. and the
fugitive ink was evacuated to produce the vascular network.
Next the HUVECs were introduced to line the channels to create
blood vessels.
FIG. 24 shows endothelial vascular channels created by the
described method. HUVECs are in evacuated GelMA gel. The channel
diameter was about 350-450 microns. Live cells shown in green were
stained with calcein; dead cells shown in red were stained with
ethidium homodimer.
FIG. 25 shows endothelial vascular channels (red cells) surrounded
by printed regions of two cell types, 10T1/2 fibroblasts, and NIH
3T3 cells, printed in a GelMA gel.
FIG. 26 shows perfused endothelial vascular channels (red cells)
that support the growth of surrounding HDNF fibroblasts (green).
The channels are created in a gel using the described method, and
the gel is embedded in a perfusion chip that facilitates the
connection of the tissue to an external peristaltic pump that
drives flow through the endothelial cell lined channels.
Example 2
Printing Endothelial Cell-Laden Fugitive Filaments
The fugitive ink, either Pluronic F-127 or gelatin contained live
HUVEC cells, as shown in FIGS. 14A-G.
Cells were first dispersed in a Pluronic-F127 ink (FIGS. 14A and
D), and then a 10% wt/v gelatin methacrylate matrix in DMEM media
containing 0.3% wt/v Irgacure 2959 photoinitiator was cast
surrounding the cells (FIGS. 14B and E) and crosslinked via UV
exposure. Next, the Pluronic F-127 ink was liquefied by cooling to
4.degree. C. and cells suspended in the ink were allowed to settle
and stick to the wall (FIGS. 14C and F). After removal of the ink
and culturing of the cells for several days, the cells remained
adhered to the walls (FIG. 14G).
Example 3
Generating Cerebral Organoids
To synthesize a cerebral organoid (shown in FIGS. 7A-I) the
following method was used.
6-well plates were coated in matrigel by incubating human
ESC-certified growth-factor depleted Matrigel, diluted in DMEM/F12
medium at the manufacturer's (Corning) batch specific recommended
concentration, for 1 h at room temperature
Human iPSCs were maintained in mTeSR medium on the matrigel coated
plates and passaged using Accutase when colonies begin to
merge.
At passaging, Human iPSCs were dissociated for 15 minutes using 1
mL Accutase reagent, then diluted in 11 mL of DMEM/F 12 medium,
centrifuged at 200 g for 5 minutes, resuspended in 1 mL of AW
medium, counted using a cellometer, and seeded at a density of
600,000 cells per well of an Aggrewell.TM. 400 plate, which
corresponds to a per-microwell density of 500 iPSCs (FIG. 7A). The
media volume was brought up to 2 mL using AW, containing 10 .mu.M
ROCK inhibitor Y-27632. The time point `day 0` corresponds to the
day that the iPSCs were first seeded in Aggrewells.TM..
After 24 hours (day 1), the iPSCs had formed embryoid bodies (FIG.
7B), and the embryoid bodies were harvested by gentle pipetting and
transferred into ultra-low adhesion plates (Corning, Inc.) and
maintained in AW medium without ROCK inhibitor for 2 days.
On day 3, embryoid bodies were transferred into NIM and cultured
for 5 days (FIGS. 7D-F) before being transferred into NDM1 (FIG.
7G).
On day 11, cerebral organoids were transferred into 1 .mu.L
droplets of Matrigel by pipetting 800 nL of ice cold Matrigel onto
a sheet of parafilm, and adding 200 nL of media containing the
organoid to the droplet. The Matrigel droplet was then maintained
at 37 C for 10 minutes for the Matrigel to gel, and then
transferred into a spinning bioreactor containing NDM1 (as shown,
e.g., in FIG. 5A).
On day 18, the flask media was changed to NDM2. The media was
replaced weekly.
Example 4
Confirming Endothelial Progenitors in Sprouting Embryoid Body
Sprouting embryoid bodies were formed by following the procedure
outlined in Example 3, but replacing the use of NIM with EGM-2
medium, and NDM1 and NDM2 media were supplemented with 100 ng/ml of
VEGF to encourage endothelial development and proliferation.
As shown in FIG. 27, left image, organoids formed using this
process were analyzed by immunofluorescence for nestin (green) (a
neural progenitor marker, and an endothelial marker), and Sox 1 (a
neural progenitor marker) (red). The strong staining of nestin, and
absence of Sox 1, as well as the tubular morphology of the cells
suggests an endothelial lineage. (FIG. 27, right image). When
stained for Sox 1 (green) and VE-Cadherin (red) (a specific
endothelial marker), the presence of neural rosettes (green
circles) in organoid body and co-presence of vasculature (red) was
detected.
Results shown in FIG. 28 further confirm the presence of cortical
structures with mature neurons.
Example 5
Confirming Presence of Neural Structures
To confirm presence of neural structures in cerebral organoids,
cerebral organoids were developed according to the protocol in
Example 3, and immunostained for Sox 1 (green) and neuron-specific
.beta.-III tubulin, also known as the clone Tuj-1 (red). As shown
in FIG. 28, the radial arrangement of neural projections confirms a
cerebral organoid phenotype.
Example 6
Generating Vascularized Cerebral Organoids from iPSCs
The following method was used to synthesize vascularized cerebral
organoids from iPSCs shown in FIGS. 29 and 30.
Vascularized cerebral organoids were prepared by the method
outlined in Example 3, but instead of using NIM, embryoid bodies
were cultured in EGM-2 medium supplemented with 1:100 N2
supplement. NDM1 and NDM2 were also supplemented with 100 ng/ml of
VEGF.
Organoids formed using this process were analyzed by
immunofluorescence for neural progenitor cells (Sox 1) and vascular
endothelial cells (CD 31) which are shown in red and green,
respectively, in the top-left image. To further study neural
structures contained within organoids cultured using this process,
we stain for N-cadherin (green, top right image) which, following
natural neural development, is shown to line the ventricular wall.
The bottom-right image is stained for (Sox 2) which identifies the
neural stem cells that form the neural rosettes that surrounds the
ventrical like structure. Both the top-right and bottom-right
images are stained with DAPI (blue) which is localized with cell
nuclei.
Example 7
Creating a Multi-Population Organoid (Approach 1)
To determine whether a multi-population organoid can be synthesized
using the `common sense` approach of mixing human umbilical-vein
endothelial cells (HUVECs) with induced pluripotent stem cells
(iPSCs), the following method was used.
iPSCs were cultured as outlined in Example 3, but prior to adding
the iPSCs into the Aggrewells.TM., cells were mixed in a 1:1 ratio
with HUVECs, then the mixture was added to the Aggrewells.TM. and
centrifuged for 3 minutes at 100 g.
As shown in FIG. 31, initially, the two types of cells dispersed in
the medium. However, at day 1, the `common sense` approach of
mixing HUVECs with iPSCs results in a `phase separation` of the two
cell types into their two distinct populations. Shown in green are
iPSCs; shown in red are HUVECs.
Example 8
Vascularizing Multi-Population Organoids
Human induced pluripotent stem cells (iPSC's) were maintained in
culture on vitronectin coated non-tissue culture treated 6-well
plates in mTeSR1 medium. To passage, cells were treated with
accutase for 5 minutes, rinsed with DMEM/F12 containing 15 mM
HEPES, and gently triturated by pipetting up and down twice with a
P1000 pipette tip. Cells were then replated in mTeSR1 medium
containing 10 .mu.M Rho-kinase inhibitor (ROCK-i). After 12-20
hours, the cells were replaced in mTeSR1 without ROCK-i, and media
was changed every day.
To form embryoid bodies, at `Day 0`, cells were passaged as
described above, except for using a longer, 15 minute accutase
treatment to separate the colonies into single cells to aid
accurate counting. Once counted, the iPSC's were added to an
Aggrewell.TM. 400 plate, at a total density of 500 cells per
micro-well, centrifuged at 100 g for 3 minutes, and cultured in 2
ml accutase containing 10 .mu.M ROCK-i to form embryoid bodies. At
this point, if a genetically modified population of iPSC's is to be
used, they can be added and mixed to the single cell suspension of
wild-type iPSC's at a defined cell-count ratio, added to the
Aggrewell.TM. plate, then centrifuged at 100 g for 3 minutes to
generate a mixed population of cells in each micro well.
After 20 hours, `Day 1`, embryoid bodies were formed in the
microwells, and the media was replaced with Aggrewell.TM. medium
without ROCK-i. Media was changed daily until `Day 3`, at which
point embryoid bodies were removed from the microwells by gentle
pipetting and cleaned by rinsing on the surface of a 40 .mu.m
reversible cell filter, before flipping the filter to release the
embryoid bodies using 5 ml of NIM supplemented with 1:100 N2
supplement. Embryoid bodies were transferred to ultra-low adhesion
6-well plates for suspension culture, and are agitated twice per
day to prevent aggregation.
After four days in suspension culture `Day 7`, cells were implanted
into a microvascular scaffold formed by either pin-casting or
molding around a sacrificial printed filament of either Pluronic
F-127 or gelatin. The matrix was comprised of matrigel mixed with
either rat rail collagen type I (2 mg/ml) or fibrinogen (10 mg/ml).
The vascular network contained two independent networks, an
`arterial` and a `venous` network. The culture medium was switched
to neural differentiation medium, phase 1, comprising a 1:1 mixture
of DMEM/F12 and Neurobasal, supplemented with 1:200 N2 supplement,
1:100 B27 supplement without vitamin A, 1:200 MEM-NEAA, 1.times.
glutamax, and 1.times. .beta.-mercaptoethanol. Next, specific
angiogenic factors were added to one or both networks to encourage
vascular sprouting. Media was pumped through the two independent
networks by means of a peristaltic pump, and media was replaced
every two days.
At `Day 11`, the media was replaced with neural differentiation
medium, phase 2, which is the same as neural differentiation
medium, phase 1, except that the B27 without vitamin A was replaced
with B27 with vitamin A.
DOX was added at 100 ng/ml to the medium (can be added at any phase
of the differentiation process) to induce the transdifferentiation
or directed differentiation of the genetically modified iPSC's.
Furthermore, a second, orthogonal signal can be added to the media
conditions described to induce sprouting of induced endothelium to
enhance the formation of a capillary plexus that connects both
venous and arterial systems. Adding an angiogenic factor can
provide a gradient to induce directional angiogenesis.
Once angiogenesis has occurred sufficiently to connect the two
networks, the positive pressure is applied, via a peristaltic pump
to only the arterial side, allowing fluid flow through the
connecting capillaries to the venous side.
As shown in FIG. 32, a fluorescent and non-fluorescent population
of iPSCs, representing a genetically modified subpopulation and a
wild-type subpopulation, respectively, can be mixed prior to adding
into Aggrewells.TM. (FIG. 32, left image). After 20 h in vitro, the
iPSCs coalesce to form an evenly distributed mixed population of
wild-type and genetically modified iPSCs.
Example 9
ETV2 Can Efficiently Induce Endothelial Phenotype
FIG. 33 shows that populations of iPSCs that have been transformed
with a doxycycline inducible promoter for a different transcription
factors can be directly-differentiated to endothelial cells with
varying degrees of efficiency when doxycycline is added to mTeSR1
medium.
Specifically, 5 separate populations of iPSCs were transformed,
using electroporation of a PiggyBac transposon system, with either
the transcription factor ETS-related gene (ERG), ETS-varient 2
(ETV2), Brachyury (T), or a combination of all four of the listed
transcription factors (All 4). FIG. 34 shows flow cytometry data
for two endothelial genes, PECAM-1 (also known as CD31) and
vascular endothelial cadherin (VECad).
In this example, doxycycline-induced overexpression of ETV2
resulted in the largest percentage of cells (.about.8%) converting
to an endothelial state after 5 days in mTeSR1 medium containing
500 ng/ml of doxycycline. The overexpression of all 4 transcription
factors also resulted in a measurable expression of vascular
markers, but was less efficient than overexpression of ETV2
alone.
FIG. 35 shows iPSCs that were transformed with a dox-inducible ETV2
vector and were cultured for 5 days in mTeSR1 containing 500 ng/ml
doxycycline resulted in a significant number of cells exhibiting an
endothelial `cobblestone` morphology that after performing an
immunofluorescence protocol, stained brightly for VECadherin (right
image, orange cells) and several co-stained with CD-31 (right
image, purple cells), while those that were cultured in mTESR1
alone remained pluripotent, as visualized by positive Oct4 staining
(left panel, green cells).
Example 10
ETV2 Overexpression can Induce Endothelium in 3D Culture
Embryoid bodies were prepared in Aggrewell.TM. plates as described
in Example 3 using ETV2 transformed iPSCs.
FIG. 36 shows a time series of phase contrast micrographs of an
embryoid body harvested from Aggrewells.TM. at day 3 and cultured
in a droplet of Matrigel bathed in neural induction medium
containing 500 ng/ml doxycycline. Visible vascular sprouts begin
protruding within 12 hours of culture.
FIG. 37 shows vascular sprouting in 10 days-cultured in matrigel
using cerebral organoid culture conditions. By adding 10 nM PMA, a
PKC-alpha activator, vascular sprouting was dramatically increased.
However, PKC also mediates neurite outgrowth, and may interfere
with cerebral organoid development.
Example 11
100% ETV2 Inducible Cells Generate Vascular Plexus
FIG. 38 shows embryoid bodies formed using a suspension of
doxycycline inducible ETV2 expressing iPSCs prepared in Matrigel as
described in Example 10. NIM, and NDM1 media were used as described
in Example 3 but supplemented with 500 ng/ml doxycycline. On day
11, embryoid bodies were fixed and stained using standard
immunofluorescence protocols.
Referring to FIG. 38, the images demonstrate the development of a
sprouting vascular plexus as indicated by the positive staining of
VE-Cadherin (red cells) with no discernable neural progenitor cells
found (as indicated by the absence of Sox 1 expressing cells).
Example 12
Long-Term Cerebral Organoid Culture
FIG. 39 shows embryoid bodies formed using a suspension of
doxycycline inducible ETV2 expressing iPSCs prepared as described
in Example 10, except that on day 1, embryoid bodies were harvested
and mixed into 2 mg/ml of rat tail collagen, type I, in AW medium
and neutralized with sodium hydroxide. The collagen solution
containing embryoid bodies was then pipetted into 6-well plates,
and incubated at 37.degree. C. to enable formation of collagen
fibrils. Next, 2 ml of AW medium was pipetted on top of the gel. On
day 3, embryoid bodies were transferred into NIM without
doxycycline (top row) or with doxycycline (bottom row), and
cultured for 5 days. On day 8, the NIM medium was replaced with
NDM1 with (bottom row) or without (top row) doxycycline. By day 10,
phase contrast imaging displayed a clear phenotypic difference
between embryoid bodies cultured with or without doxycycline. The
embryoid bodies that were cultured without doxycycline had large
neuroepithelial regions (arrows) visible in phase contrast. Those
that were cultured with doxycycline exhibited a sprouting
endothelial phenotype.
Example 13
Titrating Percentage of Wildtype vs. ETV2 Inducible Cells
Embryoid bodies were formed as described in Example 3, except that
before seeding the cells into Aggrewell.TM. plates, a mixed
suspension of wild-type iPSCs and drug inducible ETV2 cells were
mixed at a ratio of 4:1 (wild-type:ETV2). In addition, doxycycline
was added to NIM, NDM1 and NDM2 at 500 ng/ml, and cerebral
organoids were not embedded in Matrigel and were cultured in
suspension culture, without the use of the spinning flask. At day
14, the resulting vascularized cerebral organoid was fixed and
stained using standard immunofluorescence protocols.
FIG. 40 shows an immunofluorescence stained vascularized cerebral
organoid. In this organoid, there are clear regions of neural
progenitor cells, as highlighted by Sox 1 expression (purple),
followed by an outer layer of neural cells that arise from the
underlying layer of neural stem cells, as indicated by
neuron-specific .beta.III-tubulin expression (green). Furthermore,
the organoid was surrounded by a vascular plexus, as indicated by a
positive staining for VE-cadherin (red).
Example 14
Embedding Mixed Organoids in Matrigel Between Perfusable
Channels
FIG. 41 shows an organoid that underwent the following
procedure.
Embryoid bodies were formed according to the process outlined in
Example 3, except that a mixture of wildtype (60%) and
drug-inducible ETV2 cells (40%) were used to seed the
Aggrewells.TM..
On day 1 after seeding in Aggrewell.TM. plates, embryoid bodies
were harvested and placed in ultra-low adhesion plates in AW for 2
d.
On day 3, the media was changed to NIM containing 500 ng/ml
doxycycline.
On day 5, an embryoid body was injected into a Matrigel droplet,
using a method outlined in Example 10.
After gelation in an incubator for 10 minutes at 37.degree. C., the
Matrigel droplet was added to a collagen gel containing two linear
perfusable channels. The Matrigel was positioned such that they
organoid lay between the two channels. Next, the external channels
were connected to fluid reservoirs which were allowed to
gravity-feed through the channels via hydrostatic pressure, and
which was recirculated continuously via a peristaltic pump. The
organoid was grown in neural induction medium with doxycycline for
a total of 8 days and the phase contrast image was taken on day 11.
Referring to FIG. 41, vascular sprouts can be seen emerging from
the central, dense organoid and the sprouts are approaching the
adjacent perfusable channel.
Although the present invention has been described in considerable
detail with reference to certain embodiments thereof, other
embodiments are possible without departing from the present
invention. The spirit and scope of the appended claims should not
be limited, therefore, to the description of the preferred
embodiments contained herein. All embodiments that come within the
meaning of the claims, either literally or by equivalence, are
intended to be embraced therein.
Furthermore, the advantages described above are not necessarily the
only advantages of the invention, and it is not necessarily
expected that all of the described advantages will be achieved with
every embodiment of the invention.
* * * * *